Multifunctional aminoquinoline therapeutic agents

ABSTRACT

Aminoquinoline compounds useful for treating chronic pain, addiction, and other conditions are provided. The aminoquinoline compound is represented by Formula (I):in which the substituents thereof are defined as set forth in the specification.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/130,789 filed Sep. 13, 2018, issued as U.S. Pat. No. 10,435,371,which is a continuation of U.S. application Ser. No. 15/318,964 filedDec. 14, 2016, issued as U.S. Pat. No. 10,112,905, which is a Section371 US National Phase of International Application No. PCT/US2015/036473filed Jun. 18, 2015, which claims priority to U.S. Application No.62/015,152 filed Jun. 20, 2014, the disclosures of which areincorporated by reference in their entirety. This application is relatedto U.S. application Ser. No. 16,125,252 filed Sep. 7, 2018, issued asU.S. Pat. No. 10,391,088.

GOVERNMENT SUPPORT

This invention was made with government support from the NationalInstitutes of Health, Grant No. R44-AA-009930. The United Statesgovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to aminoquinoline compounds and their useas therapeutic agents. In addition, the present invention relates tomethods of treating and preventing chronic pain and treating addictionwith certain aminoquinoline derivatives.

BACKGROUND OF THE INVENTION

Pharmaceutical science is undergoing changes in its perception ofdiagnostic criteria for categorization of disease and the value ofmedications that are engineered to simultaneously bind to and affect thefunction of more than one target. For a number of years, thepharmaceutical industry has diligently followed the paradigm of singletarget-based drug discovery, but the successes with this approach forgenerating novel medication have declined drastically, leading many toquestion this approach (Csermely et al., 2005; Sams-Dodd, 2005). Morerecently, there has been significant discussion that design ofmulti-target drugs (those in which a single molecule can effectivelyinteract with more than one target in a disease-perturbed pathway) maybe a more optimal approach to discovering new and more effectivemedications (Lu et al., 2012; Pang et al., 2012). In terms ofcategorization of various disease states it has become obvious that anumber of diseases thought to be categorically different, may haveetiologically similar pathways determining the pathology. This isparticularly evident with neurodegenerative diseases where recent workindicates that cell-to-cell transmission of misfolded proteins may bethe underlying cause of several, previously thought to be different,disease states (e.g., Parkinsonism, Alzheimer's disease, Huntington'sDisease, etc.) (Guo and Lee, 2014).

In psychiatric disease areas such as schizophrenia andaffective/cognitive disorders, similar neurotransmitter pathwaysconsistently come to attention, as evidenced by the recent review on theglycine transporter (Harvey and Yee, 2013). In this review the authorsalso draw attention to the fact that the glycine receptor andtransporter systems in the central nervous system (CNS) also areinvolved in alcohol dependence, pain and epilepsy. A more directdiscussion of the similarities between alcohol dependence and chronicpain disorders can be found in a review authored by Egli, Koob andEdwards entitled “Alcohol Dependence as a Chronic Pain Disorder” (2012).The possible relationship between alcohol dependence and chronic painsyndromes is underscored by the current reports that drugs which havebeen used to treat chronic pain are now being found to be efficacious inpreventing relapse in alcohol dependent subjects. A demonstration ofthis phenomenon is the use of gabapentin (which is a T-type calciumchannel blocker) to treat chronic pain (Moore et al., 2014) andalcoholism (Mason et al., 2014).

Throughout the world millions of people suffer from chronic pain (e.g.,116 million in the U.S. alone (Institute of Medicine, 2011) and fromalcohol abuse and dependence (e.g., 18 million in the U.S. (Grant etal., 2004)) or both, and similarities in the neurochemistry subsumingboth chronic pain syndromes and alcohol dependence go well beyond thecommon involvement of T-type calcium channels. Three neurochemicalsystems have, in particular, been linked to both chronic pain andalcohol dependence. These systems are the GABA, cannabinoid and theopioid transmitter systems in brain and spinal cord (Zeilhofer et al.,2012). The GABA-A receptor system has been linked to both the acute andchronic actions of ethanol including the development of dependence andthe generation of craving during periods of abstinence (Enoch et al.,2013; Kumar et al., 2004; Tabakoff and Hoffman, 2013). Chronic ethanolconsumption by mice upregulates (increases expression of) delta opiatereceptors in the CNS (van Rijn et al., 2012), and delta opiate receptorexpression and function in certain areas of brain (e.g., ventraltegmental area) has been shown to modulate ethanol consumption byanimals (Clapp et al., 2008). Some of the effects of agents which act asagonists at the delta opioid receptors have been proposed to be mediatedvia modulation of GABA neuron function (Kang-Park et al., 2007). Themost informative recent description of how delta opiate receptors canmodulate GABA-A receptor function is contained in (Margolis et al.,2011) and in that report, it is stressed that increases in deltareceptor function “only appear following challenges such asinflammation, stress and administration of rewarding (addicting) drugs”,and that such increases in function can change activity of otherneurotransmitter systems (e.g., GABA).

The upregulation of delta opiate receptors is also a definitive aspectof the development of chronic pain (Cahill et al., 2003) and mice withgenetic deletion of the delta opiate receptor are inherently moresensitive to painful stimuli (Gaveriaux-Ruff et al., 2011). Although itis rational to consider that delta opiate receptors are good targets forpharmaceuticals to treat chronic pain, there are currently no deltaopiate receptor selective drugs approved by the Food and DrugAdministration and some candidates have failed in Phase II clinicaltrials (van Rijn et al., 2013). A confound in the simplistic view thatdelta opiate receptors in and of themselves can reduce chronic pain, isthat tolerance rapidly develops to the antihyperalgesic actions of deltaopiate receptor agonists. Part of this “tolerance” mechanism is mediatedvia increased inhibition of GABA release in spinal cord and brain stemby upregulated delta opiate receptors which arise during development ofchronic pain (Taylor, 2009; Zhang et al., 2006).

There also has been discussion in the literature that one shouldconsider the possibility of preventing the development of chronic painby medications administered in the early phase of the neuropathologicalprocess that produces chronic (neuropathic) pain syndromes (Kehlet etal., 2006; Van de Ven and John Hsia, 2012). One of the more acceptedmechanisms for the progression of acute injury to chronic pain is anupregulation of voltage sensitive sodium channels (e.g., Na_(v)1.7 andNa_(v)1.8) in sensory neurons (Belkouch et al., 2014; Strickland et al.,2008). Studies performed with endogenous delta receptor agonists such asenkephalin have indicated that activation of delta opiate receptors canprevent the upregulation of Na_(v)1.7 in sensory neurons in rats treatedto produce diabetic neuropathy (Chattopadhyay et al., 2008).Interestingly, the transfection of neurons with a vector, resulting in aconstant release of GABA, also prevented the pathologic increase inNa_(v)1.7 resulting from chronic hyperglycemia (diabetes) (Chattopadhyayet al., 2011). Additionally, there is evidence that delta opiatereceptors present in Na_(v)1.8-expressing nociceptive sensory neuronsplay a critical role in pain mechanisms (Gaveriaux-Ruff et al., 2011).Thus one can postulate that a novel medication that can activate bothGABA receptors and delta opiate receptors may prevent the development ofchronic (neuropathic) pain syndromes by interfering with the pathologyinduced upregulation of Na_(v)1.7 and 1.8 channels and/or othermechanisms. A medication that can simultaneously activate GABA receptors(Zeilhofer et al., 2012) and activate delta opiate receptors, as well asinhibiting the Na_(v) 1.7 and 1.8 channels, can also be of benefit inreducing pain even after the development of a chronic pain syndrome.

The cannabinoid neurotransmitter system of the brain and spinal cord, inmany ways resembles the opioid transmitter system. The endogenousagonists for cannabinoid and opiate receptors differ (i.e., anandamideis the agonist at the cannabinoid (CB1) receptors, while enkephalins arethe agonists at the delta opiate receptors), but the receptorcharacteristics and physiologic function of the cannabinoid (CB1)receptor and the delta opiate receptor are quite similar. Both are Gprotein coupled receptors (GPCRs) that signal through the G_(i)/G_(o)proteins and affect the function of the same set of neuronal enzymes andchannels which carry out the CB1 and delta opioid receptor effects(Howlett et al., 2002). Both CB1 receptors and delta opiate receptorshave been designated as targets for control of chronic pain syndromes(Normandin et al., 2013; Pernia-Andrade et al., 2009), and for reducingcraving and high levels of alcohol consumption in alcohol dependentanimals (Femenia et al., 2010; van Rijn et al., 2010). It is notablethat pharmacological and direct interactions between delta opiatereceptors have also been noted (Manzanares et al., 1999; Vigano et al.,2005). Particularly in the control of pain, delta 9-tetrahydrocannabinol(THC, a CB1 receptor agonist) has been shown to have synergistic effectswith opiates, and these effects of THC have been stated to result fromits actions at the delta opiate receptor, as well as CB1 receptors(Cichewicz, 2004). A more current theory of CB1 receptor and deltaopiate receptor interactions is that there is a physical interaction(heterodimerization) between these receptors (Rios et al., 2006).Therefore, medications that affect the function of the CB1 cannabinoidreceptor or the delta opiate receptor can modulate the activity of theinteracting partner receptor system and have similar end effects on GABArelease (Olive, 2010).

A joint, beneficial effect of multi-modal medications may as well beseen in preventing and treating relapse in alcoholics. By simultaneouslyenhancing GABA-A receptor function during a period of alcohol withdrawalin an alcohol dependent animal and modulating of cannabinoid or deltaopiate receptors in a dependent subject (Bie et al., 2009a), control ofcraving and a reduction of relapse can be achieved.

The recent revision (the fifth) of the Diagnostic and Statistical Manualof the American Psychiatric Association (DSM V) defines alcoholism(Alcohol Use Disorder, AUD) by eleven criteria, of which two have to bemet during the same 12 month period for an individual to be diagnosed assuffering from AUD (NIH Publication No. 13-7999, 2013). A novel additionto DSM V is a criterion of craving to the list of criteria that candefine AUD. Craving was not a component of earlier DSM diagnosticcriteria, but over recent years, the concept and phenomenon of cravinghas become a primary reason for individuals to relapse to alcohol useafter a period of sobriety (Anton, 1999). Craving in the context of DSMV, is distinguished from attempts by an individual to control withdrawalsigns which occur early (within a day or two) after an individual stopsconsuming ethanol, and alcohol or a closely related substance such asbenzodiazepines may be taken to relieve withdrawal signs. The overtsigns of alcohol withdrawal in humans last for five days to a week, andin terms of treatment, constitute the detoxification stage of treatingalcoholism. The manifestations of craving as defined by DSM V and inother publications (Kavanagh et al., 2013) are cognitive-emotionalevents over a period of years rather than days, and are manifestationsof limbic system function (Heinz et al., 2009). The early withdrawalsigns are a neural hyperactivity syndrome exhibited over most of thebrain with particular involvement of the cerebral cortex(Coutin-Churchman et al., 2006). One of the most significantdistinctions between the biologic characteristics of the withdrawalsyndrome and the later manifestations of craving is in terms ofpharmacologic treatments. For instance, benzodiazepines are commonlyused to advantage in treating the acute stages of the alcohol withdrawalsyndrome, but are contraindicated for more prolonged use in allaycraving (Licata and Rowlett, 2008). On the other hand, the drugs mostcurrently used in the U.S.A. for treating craving and preventingrelapse, i.e., acamprosate and naltrexone, are significantly moreeffective if given after detoxification or after a prescribed period(weeks) of abstinence.

There is an ongoing need for medications that can treat chronic pain,addiction, addiction relapse, and the like. Methods of synthesis ofsubstituted quinolone ureas are disclosed herein, which significantlyexpands on the series of chemical entities which have substitutions onthe terminal nitrogen of 4-ureido-5,7-dihalo-2-carboxy-quinolines and inwhich the 2-position is a carboxy group, an ester, a ketone, an ether oran amide. Certain of the compounds synthesized by the methods describedherein unexpectedly have affinity for and pharmacological actions at themammalian GABA-A receptor, the cannabinoid (CB1) receptor, the voltagesensitive sodium channels (Na_(v) 1.7 and 1.8) and/or the delta opiatereceptor while having little or no affinity for a large number of otherreceptors/channels/enzymes.

GABA-A receptors are agonist gated ion channels which respond to thepresence of the neurotransmitter GABA by increasing permeability tochloride ions and thus generating hyperpolarization and inhibition ofongoing activity in neurons. The GABA-A receptors are composed of fivedistinct protein subunits with the majority of the GABA-A receptors inbrain having a composition consisting of two alpha (α) subunits, twobeta (β) subunits and one gamma (γ), delta (δ), theta (θ) or pi (π)subunit. Currently 19 GABA-A receptor subunits are known (α 1-6, β 1-3,γ 1-3, δ, ε, θ, π and 3 rho (ρ) subunits).

GABA-A receptors are the site of action of a number of clinicallyimportant drugs such as benzodiazepines, barbiturates, anesthetics,analeptics, neuroactive steroids, etc. and all of these drugs interactwith binding sites that are partially or completely distinct from oneanother. These binding sites, including the binding sites for GABAitself, are generated by the interactions of various subunits and areformed at the interface of the various subunits that come together toproduce the pentameric combination that characterizes the native GABA-Areceptors in brain and spinal cord. Given the large number of subunitcombinations that are possible (although not all have been demonstrated)it is not surprising that the GABA-A receptor demonstrates a complexpharmacologic profile.

A large number of molecules have been synthesized and shown to interactwith GABA-A receptors of a particular subunit combination, and thesemolecules display a variety of pharmacologic characteristics includingsedation, anesthesia, anxiolysis, anticonvulsant effects, musclerelaxation, analgesia, antipsychotic actions and even modulation ofimmune system function depending on the subunits present in particularGABA-A receptors and the way in which the subunits interact.

The most common type of GABA-A receptor present in brain consists of twoα₁ subunits, two β₂ subunits and a γ₂ subunit and these receptors aremostly located within the membrane of the post-synaptic neuron at thesynapse. These and other synaptically located GABA-A receptors aremediators of phasic GABA signals in response to GABA release from thepre-synaptic terminal. On the other hand, GABA-A receptors that containa δ subunit instead of a γ subunit are located extra-synaptically(outside of the synapse) and generate a tonic inhibition of thepost-synaptic neuron in response to GABA that “leaks” out of thesynaptic cleft. These extra-synaptic GABA-A receptors, not only aredistinguished by their δ subunit but also by a subunits. Theextra-synaptic GABA-A receptors can contain only α₁, α₄ or α6 subunitsin association with the δ subunit.

The α₁ subunit in association with the δ subunit is primarily found inthe interneurons and pyramidal cells of the hippocampus, the α₄ togetherwith δ is expressed in the thalamic relay neurons and dentate gyrusgranule cells and the α₆ and δ subunit containing GABA-A receptors areprimarily localized to the cerebellar granule cells. The differentanatomical distribution of particular a subunits in conjunction with theδ subunit bespeaks different function and further physiologicdifferences exist between the different α and δ subunit combinations.For instance, the extra-synaptic GABA-A receptors composed of the α₁, β₂or β₃, and δ subunits have very low permeability for chloride ions evenin the presence of high concentrations of GABA. On the other hand, these“silent” receptors are quite active when GABA is applied together with apositive allosteric modulator which is effective at δ subunit containingGABA-A receptors. It is therefore clear that discovery of molecularentities that have selectivity for particular subunit combinationswithin the GABA-A receptor can generate novel medications either withpredicted or unanticipated spectra of physiologic or anti-pathologiceffects.

Given the above described literature on the involvement of GABA-A, CB1and delta opiate receptors in relapse drinking in alcohol dependentsubjects and in the etiology of chronic pain syndromes, a series of thecompounds described herein were tested in animal models of relapsedrinking and chronic pain syndromes. A pattern of effects was observedthat can predict that particularN-substituted-4-ureido-5,7-dichloro-2-carboxy (orcarboxyester)-quinolines can be effective in preventing relapse inaddicted humans and also can be effective in treatment and prevention ofchronic neuropathic pain in humans.

SUMMARY OF THE INVENTION

The present invention provides multifunctional aminoquinoline compoundsthat are useful for treatment of one or more conditions such as alcoholor drug dependence, addiction relapse, chronic pain, and otherconditions that can benefit from activation of GABA-A, CB1, and deltaopiate receptors and inhibition of voltage sensitive sodium channels(Na_(v), 1.7 and 1.8).

In a first embodiment, aminoquinoline compound is represented by Formula(I):

in which the substituents thereof are defined as follows: R¹ is H, C₂-C₄alkyl, C₂-C₄ alkenyl, halo, Z¹R⁹, or N(R¹⁰)(R¹¹). R² is H, C₁-C₄ alkyl,C₂-C₄ alkenyl, halo, Z²R¹², N(R¹³)(R¹⁴), or C₁-C₄ alkyl substituted withone or more moiety selected from the group consisting of C₁-C₄ alkyl,C₂-C₄ alkenyl, halo, Z³R¹⁵, N(R¹⁶)(R¹⁷); each R³, R⁴, R⁵, and R⁶independently is H, C₁-C₄ alkyl, C₂-C₄ alkenyl, halo, Z³R¹⁸, orN(R¹⁹)(R²⁰); X¹ is N or CH; each R⁷ and R⁸ independently is H, C₁-C₆alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, aryl, or C₁-C₆ alkyl substitutedwith one or more moiety selected from the group consisting of C₁-C₄alkyl, C₂-C₄ alkenyl, nitro, halo, Z⁴R²¹, and N(R²²)(R²³); or R⁷ and R⁸together with X¹ form a 5 to 8 member saturated, unsaturated, oraromatic organic cyclic or heterocyclic moiety; each R⁹, R¹⁰, R¹¹, R¹²,R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², and R²³ independentlyis H, C₁-C₄ alkyl, or C₁-C₄ alkyl substituted with one or more moietyselected from the group consisting of C₁-C₄ alkyl, C₂-C₄ alkenyl, C₂-C₄alkynyl, halo, heteroaryl, Z⁵R²⁴, and N(R²⁵)(R²⁶). Each of Z¹, Z², Z³,Z⁴, and Z⁵ independently is O, S, NH, C(═O)O, O—C(═O), C(═O), orC(═O)NH. Each R²⁴, R²⁵, and R²⁶ independently is C₁-C₄ alkyl with theproviso that when R¹ is Z¹R⁹, Z¹ is C(═O)O, R⁹ is H or C₁-C₂ alkyl, eachof R³ and R⁵ is halo, X¹ is N, and each of R⁴ and R⁶ is H, then at leastone of R⁷ and R⁸ is not a phenyl, alkoxy-substituted phenyl, or C₁-C₆alkyl group.

In a second embodiment, the aminoquinoline compound is a compound ofFormula (II):

wherein X¹, R¹, R⁷ and R⁸ are as defined in Formula (I) above, and eachX² and X³ independently is an electron withdrawing group such as halo,nitro, and the like, with the proviso that when R¹ is Z¹R⁹, Z¹ is C(═O)Oor C(═O), R⁹ is H or C₁-C₄ alkyl, and X¹ is N, then at least one of R⁷and R⁸ is not a phenyl or alkoxy-substituted group.

In a third embodiment, the aminoquinoline compound is anaminodihaloquinoline compound of Formula (III):

wherein X² and X³ each independently is halo, and each of X¹, R¹, R⁷, R⁸and R⁹ are as defined in Formulas (I) and (II) described above, with theproviso that when R⁹ is H or C₁-C₂ alkyl, and X¹ is N, then at least oneof R⁷ and R⁸ is not a phenyl or alkoxy-substituted phenyl group.

In a fourth embodiment, the aminoquinoline compound is a compound ofFormula (IV):

wherein each of X², X³, R¹, R⁷, and R⁸ are as defined in Formulas (I)and (II) above.

In a fifth embodiment, the aminoquinoline compound is anaminodihaloquinoline compound of Formula (V):

wherein each of X², X³, R⁷, R⁸ and R⁹ are as defined in Formulas (I) and(II) above.

In a sixth embodiment, the aminoquinoline compound is represented byFormula (VI):

wherein R⁷ is alkyl (preferably a 3 to 6 carbon alkyl), cycloalkyl(preferably a 3 to 6 carbon cycloalkyl), aminoalkyl or phenyl; R⁸ is H,alkyl (preferably a 3 to 6 carbon alkyl), cycloalkyl (preferably a 3 to6 carbon cycloalkyl), aminoalkyl, or phenyl; E¹ is —C(═O)OR⁹, —C(═O)R⁹,—C(═O)N(R⁹)₂, and —[C(R⁹)₂]_(n)—OR⁹; “n” is 1, 2, 3, or 4; each R⁹independently is H, C₁-C₄ alkyl, or C₁-C₄ alkyl substituted with one ormore moiety selected from the group consisting of C₁-C₄ alkyl, C₂-C₄alkenyl, C₂-C₄ alkynyl, halo, heteroaryl, Z⁵R²⁴, and N(R²⁵)(R²⁶); Z⁵ isO, S, C(═O)O or O—C(═O); each R²⁴, R²⁵, and R²⁶ independently is C₁-C₄alkyl the alkyl; each X² and X³ independently is an electron withdrawinggroup (preferably halogen or nitro); the alkyl, cycloalkyl, amino alkyl,and phenyl groups can be unsubstituted or substituted one or more timeswith an alkyl (1-3 carbons) group or an alkyloxy group (e.g., a 1 to 3carbon alkyl or alkoxy group); and when acidic or basic functionalgroups are present, the compound can be in the free acid form, free baseform, or can be a pharmacologically acceptable addition salt. When E¹ isC(═O)OR⁹, at least one of R⁷ and R⁸ is not phenyl. Preferably R⁹ is H or1 to 3 carbon alkyl. In some preferred embodiments of the compounds ofFormula (VI), E¹ is C(═O)OR⁹ and R⁹ preferably is H or 1 to 4 carbonalkyl or cycloalkyl.

Administration of the compounds of Formula (VI) can be by oral,subcutaneous, intravenous, intramuscular, intraperitoneal, transdermalor buccal routes for therapeutic treatment.

Non-limiting examples of compounds of the general Formula (VI) arederivatives of the 2-carboxy-quinolines, e.g., (N,N-dibutyl)-4-ureido-5,7-dichloro-2-carboxy-quinoline (BCUKA)N,N-diphenyl-4-ureido-5,7-dichloro-2-butanone, quinoline and the like.These compounds demonstrate affinity for the GABA-A receptor, the CB1cannabinoid receptor and the delta opiate receptor.

The di-substituted-4-ureido-5,7-dichloro-2-carboxy-quinoline and2-butanone-quinoline compounds of Formula (VI) and their methyl andethyl esters possess affinity for GABA-A, cannabinoid CB1, and deltaopiate receptors. These compounds can also act as positive allostericmodifiers of GABA action at the GABA-A receptor and as partial agonistsat the cannabinoid CB1 receptors. These compounds possess beneficialactivity in treating chronic pain syndromes arising from inflammatoryand mechanical damage to peripheral nerves with particular compoundsbeing more effective in ameliorating mechanical allodynia/hyperalgesia,while other derivatives more effectively ameliorate thermalhyperalgesia. Additionally, these compounds have dose related activityin reducing or completely blocking abstinence-induced craving andreducing escalated ethanol consumption by animals which are madedependent on ethanol.

In some embodiments, the aminoquinoline compound of Formula (I), (II),(III), (IV) (V), or (VI) is administered to the subject in conjunctionwith an additional therapeutic agent different from the aminoquinolinecompound, e.g. opiates, opiate agonists, or opiate antagonists.

In another aspect, the present invention provides a method for thetreatment of chronic pain (e.g., chronic neuropathic pain, such asperipheral neuropathic pain). The method comprises administering anaminoquinoline compound to a subject suffering from chronic pain,wherein the aminoquinoline compound is a compound of Formula (I), (II),(III) (IV), (V) or (VI) as defined above, with the proviso for compoundsof Formula (I), (II) and (III) that when R¹ is Z¹R⁹, Z¹ is C(═O)O, R⁹ isH or C₁-C₂ alkyl, each of R³, R⁵, X², and X³ is halogen, X¹ is N, andeach of R⁴ and R⁶ is H, then at least one of R⁷ and R⁸ is not a phenylor alkoxy-substituted phenyl group.

In another aspect, the present invention also provides pharmaceuticalcompositions, e.g., for treating chronic pain and of addiction relapse,comprising an aminoquinoline compound of Formula (I), (II), (III) (IV),(V), or (VI) as defined herein in combination with a pharmaceuticallyacceptable carrier. In the case of pharmaceutical compositions fortreating chronic pain (e.g., neuropathic pain) with the compounds ofFormula (I), (II) or (III), when R¹ is Z¹R⁹, Z¹ is C(═O)O, R⁹ is H orC₁-C₂ alkyl, each of R³, R⁵, X², and X³ is halogen, X¹ is N, and each ofR⁴ and R⁶ is H, then at least one of R⁷ and R⁸ is not a phenyl oralkoxy-substituted phenyl group.

In another aspect a method of preparing a compound of Formula (VI) isprovided, which comprises amidation of a5,7-dichloroquinolone-2-carboxylate intermediate (e.g., obtainable byMichael addition of 3,5-dichloroaniline to dimethyl acetylenedicarboxylate, followed by thermal cyclization of the resultant arylmaleate) with chlorosulfonyl isocyanate to generate(4-amino)-5,7-dichloro-2-carboxy-quinoline ethyl ester (a keyintermediate), which can be functionalized through reactions withrelevant electrophiles. For preparation of monosubstituted ureas, areactive urea intermediate is prepared through the reaction of a primaryamine with carbonyldiimidazole. Reacting the resulting imidazoleureawith the amino-5,7-dichloro-2-carboxy-quinoline ethyl ester in thepresence of sodium hydroxide yields a target mono-substituted urea atthe 4 position of the quinoline, with concomitant ester hydrolysis.Removal of a protecting group, such as a tert-butoxycarbonyl(BOC)-protecting group, if such is used in the synthesis of the reactiveurea intermediate, can be achieved with trifluoroacetic acid (TFA), toproduce a desired TFA salt.

For preparation of disubstituted urea derivatives, the(4-amino)-5,7-dichloro-2-carboxy-quinoline methyl or ethyl ester isacetylated at the 4-amino position with a disubstituted carbamoylchloride to form a (N,N-disubstituted)-4-ureido-5,7-dichloro-2-carboxy-quinoline ester.Optionally, the (N,N-disubstituted)-4-ureido-5,7-dichloro-2-carboxy-quinoline-ester can behydrolyzed to an (N, N-di substituted)-4-ureido-5,7-dichloro-2-carboxy-quinoline.

For the preparation of compounds in which R¹ is an amide (i.e.,C(═O)NHR⁹), the corresponding carboxylic acid (i.e., where R¹ isC(═O)OH) can be amidated by any convenient amidation reaction, e.g., byconversion of the carboxylic acid to an activated carbonyl (e.g., anacid chloride or through the use of a coupling agent such asN,N-dicyclohexylcarbodiimide, DCC) and subsequent reaction with anamine.

For the preparation of compounds in which R¹ is a ketone (i.e.,C(═O)R⁹), the corresponding carboxylic acid (i.e., where R¹ is C(═O)OH)can be amidated with N,O-dimethylhydroxylamine by any convenientamidation reaction, as described above, and subsequent reaction of theresulting N-methoxyamide a Grignard reagent to form the ketone.

In another aspect, a method of allosterically modulating the activity ofGABA-A receptors is provided, which comprises contacting the receptorwith a compound having the formula:(N,N-dibutyl)-4-ureido-5,7-dichloro-2 carboxy quinoline (BCUKA),N,N-diphenyl-4-ureido-5,7-dichloro-2-butanone quinoline (DCVK-butanone),and the like.

In another aspect, a method of directly activating the delta opiatereceptor is provided, which comprises contacting the receptor with DCUKAand/or BCUKA.

In another aspect, a method of directly activating the cannabinoid (CB1)receptor is provided, which comprises contacting the receptor withDCUK-OEt.

In another aspect, a method for treating a drug and/or alcohol-dependentindividual to prevent relapse is provided, which comprises administeringto a patient in need of such treatment an effective amount of DCUK-OEt,DCUKA, DCUK-butanone(3-(2-Butyryl-5,7-dichloroquinolin-4-yl)-1,1-diphenylurea) or aDCUK-amide (e.g.,5,7-dichloro-4-(3,3-diphenylureido)-N-ethylquinoline-2-carboxamide, or5,7-dichloro-4-(3,3-diphenylureido)-N-isopropylquinoline-2-carboxamide).

In another aspect, a method for treating a patient suffering fromchronic pain (neuropathic pain) and ameliorating such pain is provided,which comprises administering an effective amount of DCUKA, BCUKA and/orDCUK-OEt.

In another aspect, a method of preventing the development of chronic(neuropathic) pain after an individual sustains an injury or suffersfrom chemically or pathologically-induced nerve damage is provided,which comprises administering an effective amount of DCUKA and/or BCUKAto the individual prior to the time that chronic (neuropathic) pain isevident.

In another aspect, a method for potentiating the effects of otheranalgesic compounds, such as aspirin and opiates (e.g., morphine), inpatients suffering from chronic pain is provided, which comprisesadministering, together with subtherapeutic doses of aspirin or opiate,an effective amount of DCUKA and/or DCUK-OEt.

Advantageously, DCUKA, BCUKA and DCUK-OEt each exhibit affinity (Ki<10μM) for at least two of the following receptors: the GABA-A receptor,the delta opiate receptor, the cannabinoid (CB1) receptor, and thevoltage sensitive sodium channels (Na_(v) 1.7 and 1.8).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the agonist properties of DCUK-OEt in cellstransfected with the cannabinoid CB1 receptor. These cells also expressdopamine (D1) receptors and Type 7 adenylyl cyclase; both thecombination of dopamine (DA) and ACEA, and the combination ofDA/ACEA/DCUK-OEt, provided statistically significant (p<0.05) increasesin cAMP accumulation compared to DA alone.

FIG. 2 illustrates the agonist properties of BCUKA in cells transfectedwith delta opiate receptors. These cells also express dopamine (D1)receptors and Type 7 adenylyl cyclase.

FIG. 3 illustrates the agonist properties of DCUKA in cells transfectedwith delta opiate receptors. These cells also express dopamine (D1)receptors and Type 5 adenylyl cyclase. HeLa cells were transfected withthe dopamine D1 receptor, DOR and AC5 isoform. The assay included LM DA,and DCUKA concentrations ranging from 0.25-20 μM.

FIGS. 4A-4D provide experimental data on DCUKA and DCUK-Oet. FIG. 4Acompares the effects of DCUKA and DCUK-OEt alone or in the presence ofGABA on chloride currents generated by activation of GABA-A receptors.DCUKA and DCUK-OEt had no effect on chloride currents by themselves, butsignificantly enhanced GABA-induced currents. The results indicate thatDCUKA and DCUK-OEt are allosteric modulators of the actions of GABA.FIG. 4B demonstrates that the DCUK compounds are not acting by bindingto the benzodiazepine site on the GABA-A receptor since the specificbenzodiazepine antagonist (flumanzenil) had no significant effect on thepotentiation of the GABA response either by DCUK-OEt or DCUKA. FIG. 4Cillustrates the structure/activity relationship on the efficacy of fiveDCUK derivatives. This data indicates that DCUK-OEt and DCUK-butanonewere most efficacious and suggests that an ester or amide substituent atthe 2-position of DCUK is important for the positive allostericproperties of the DCUK compounds. FIG. 4D provides a graphicrepresentation of a rank ordering of the effects of DCUK-OEt on GABA-Areceptors having different subunit compositions, beginning with the mostresponsive receptor.

FIG. 5 graphically illustrates the reversal of cisplatin-inducedneuropathic pain by DCUKA (50 mg/kg). In rats treated with the cancerchemotherapy agent, cisplatin, the cisplatin treatment reduces themechanical pain threshold and DCUKA treatment reverses this effect andincreases the mechanical pain threshold to control levels. The data showthe ratio of the mechanical pain threshold after cisplatin or cisplatinplus DCUKA treatment to the pre-cisplatin treatment mechanical painthreshold. Combined analysis of the effect of 50 mg/kg DCUKA onmechanical pain threshold in a Cisplatin-induced pain model. Treatmentmeans+/−1 SEM plotted. *P-value <0.0001 compared to control (0 mg/kgDCUKA).

FIG. 6 graphically illustrates a comparison of the effects of equimolardoses of DCUKA, BCUKA and gabapentin to reverse cisplatin-inducedneuropathic pain, measured by changes in the mechanical pain threshold.Mechanical pain threshold for each animal is represented by the ratio tobaseline. The mean mechanical pain threshold±1 standard error is plottedfor each treatment group and time. *p<0.05 compared to the correspondingpre-treatment group.

FIG. 7 graphically illustrates the reversal by DCUKA (50 mg/kg) ofneuropathic pain induced by treatment of rats with Complete Freund'sAdjuvant (CFA). CFA treatment induces inflammation and reduces themechanical pain threshold. DCUKA treatment reverses the drop in themechanical pain threshold in CFA-treated rats and returns the thresholdtoward the baseline level. The data show the ratio of the mechanicalpain threshold after CFA or CFA plus DCUKA treatment to the mechanicalpain threshold prior to CFA treatment. A post-hoc Fisher's LSD t-testfor pairwise comparisons showed a significant (*) difference between 50mg/kg DCUKA and 0 mg/kg DCUKA (p-value <0.0001).

FIG. 8 illustrates a comparison of the effect of DCUKA (50 mg/kg) andBCUKA (50 mg/kg) to reverse neuropathic pain induced by treatment ofrats with CFA. The data show the ratio of the mechanical pain thresholdafter CFA treatment, or CFA plus DCUKA or BCUKA treatment, to thebaseline (pre-CFA) mechanical pain threshold. Mechanical pain thresholdfor each animal is represented by the ration to baseline for theinjected paw only. The mean mechanical pain threshold ±1 standard erroris plotted for each treatment group. *P<0.05 compared to vehicle.

FIG. 9 shows the results of a meta analysis of experiments to determinethe dose dependent effect of DCUKA to reverse CFA-induced neuropathicpain. Mechanical pain threshold for each animal is represented by theratio to baseline for the injected paw only. The mean mechanical painthreshold±1 standard error is plotted for each treatment group. This isbased on treatment means from the 5 studies included in the metaanalysis. *P<0.05 compared to the vehicle (0 mg/kg) treatment group.

FIG. 10 graphically illustrates the reversal by DCUKA (50 mg/kg) of paincaused by diabetic neuropathy. Rats were treated with streptozotocin(STZ) to induce diabetes, which reduced the mechanical pain threshold incomparison to baseline (pre-STZ treatment). DCUKA treatment reversed themechanical pain threshold to baseline. The data show the ratio of themechanical pain threshold after STZ or STZ plus DCUKA treatment to thepre-STZ mechanical pain threshold. A post-hoc Fisher's LSD t-test forpairwise comparisons, showed a significant (*) difference between 50mg/kg DCUKA and 0 mg/kg DCUKA (difference=0.60, p-value <0.0001).

FIG. 11 shows the results of a meta-analysis of experiments to determinethe dose-dependent effect of DCUKA to reverse STZ-induced neuropathicpain. Data are the ratio of the mechanical pain threshold measured afterDCUKA treatment to the baseline mechanical pain threshold measured onthe same paw. *P<0.05 compared to the vehicle treatment group.

FIG. 12 shows that the ability of DCUKA to reverse CFA-inducedneuropathic pain is reversed by the delta opiate receptor antagonist,naltrindole, which is injected into the inflamed paw. Data are presentedas mean±SEM percent of baseline paw withdrawal threshold. P-values arecompared to the DCUKA/Saline group (one way ANOVA and post hocStudent-Newman-Keuls test). Nal, Naltrindole.

FIG. 13 illustrates graphically that administration of DCUKA followingCFA injection prevents the development of CFA-induced neuropathic pain,measured by changes in the mechanical pain threshold. Note: Student'st-test is used to compare the pain threshold between group, and pairedt-test is used to compare the pain threshold within group.

FIG. 14 illustrates graphically that administration of DCUKAsimultaneously with the cancer chemotherapy agent cisplatin prevents thedevelopment of cisplatin-induced neuropathic pain, measured by changesin the mechanical pain threshold. Two way repeated measure ANOVArevealed a statistically significant difference (P=0.023) between thetwo treatments, DCUKA and vehicle. The individual p values shown in thegraph are for DCUKA versus vehicle and are calculated by the multiplecomparison method. There are also significant differences detected inthe pain threshold in the cisplatin and vehicle group (day 15, 19, 25,32) compared to day 0, before cisplatin, but no significant differencesin pain threshold between the cisplatin and DCUKA group compared to theDay 0 values (prior to cisplatin injection).

FIG. 15 shows graphically that administration of DCUKA or DCUK-OEtenhances the ability of morphine to reverse CFA-induced neuropathicpain, measured by changes in paw withdrawal threshold in a test ofthermal hyperalgesia; DCUKA-OEt significantly decreased the half-maximaleffective dose of morphine needed to reverse pain. DCUK-OEt decreasedthe ED50 for the anti-hyperalgesic actions of morphine at 18:1(†P<0.05). DCUK-OEt/morphine dose ratios compared to Morphine alone byone-way ANOVA with post-hoc Dunnett's test).

FIG. 16 illustrates that DCUKA enhances the ability of aspirin toreverse STZ-induced neuropathic pain (diabetic neuropathy). *p<0.05compared to control group (no STZ treatment). # NS compared to vehicletreatment group. **NS compared to no STZ treatment.

FIG. 17 illustrates the reduction in abstinence-induced alcoholconsumption (relapse) by DCUK-OEt in animals made physically dependenton alcohol by long term ethanol consumption.

FIG. 18 illustrates the effects of DCUK-OEt and DCUKA in an operantresponding paradigm on alcohol self-administration in ethanol dependentrats, demonstrating that DCUKA has no significant effect compared tobaseline (BSL) in ethanol vapor-exposed alcohol dependent rats, whileDCUK-OEt at 20 mg/kg brought responding level of the ethanolvapor-exposed rats down to the same level of self-administrationnon-ethanol pretreated control rats. No effect of DCUK-OEt was observedlever pressing for water by the alcohol dependent rats.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides multifunctional aminoquinoline compoundsthat activate the delta opioid receptor, cannabinoid CB1 receptor, andGABA-A (i.e., act as a positive allosteric modulator, PAM, and inhibitvoltage sensitive sodium channels Na_(v) 1.7 and 1.8). Compounds havingsuch properties provide significant opportunities for treatment ofchronic (neuropathic) pain syndrome and prevention of relapse inpatients recovering from alcohol and/or drug addiction.

Some methods described herein comprise treating a subject (e.g., a humanor animal patient) with the aminoquinoline compound (e.g., in apharmaceutical composition as described herein). The compound isadministered to the patient at a dosage and in a dosage form suitablefor the intended use and administration, as described herein. Theaminoquinoline compounds and pharmaceutical compositions comprising thecompounds can be administered as a stand-alone treatment or, optionally,the compounds and compositions can be administered in conjunction with(i.e., in combination with or concurrently with) additional therapeuticagents. For example, in the case of treating chronic pain, theaminoquinoline compounds and compositions comprising the compounds canbe administered in conjunction with an analgesic or anti-inflammatoryagent which is different from the aminoquinoline compound, e.g., anopiate, an opioid agonist, or antagonist, a non-steroidalanti-inflammatory drug (NSAID), a corticosteroid, a serotonin reuptakeinhibitor (SSRI), a calcium channel blocker (CCB), an anticonvulsant, atherapeutic agent for treating a disease (e.g., a disease associatedwith chronic pain, such as arthritis), and the like. Some specificcombinations include, for example combinations of a compound of Formula(I), (II), (III), (IV), (V) and/or (VI) with an NSAID such as aspirin,ibuprofen, or naproxen; with an SSRI such as duloxetine (e.g., CYMBALTA®brand SSRI) or fluoxetine (PROZAC® brand SSRI); with a CCB such asgabapentin or clonidine; or with an opiate or opioid such as morphine oroxycodone; or with an anticonvulsant such as pregabalin (e.g., LYRICA®brand anticonvulsant). In the case of preventing relapse in alcoholaddicted individuals, the aminoquinoline compounds and compositionscontaining same can be used with other active agents used for thetreatment of alcoholism such as opiate antagonists, anticonvulsants,antidepressants, chronic pain medications, and the like.

The aminoquinoline compounds of Formula (I), (II), (III), (IV), (V) and(VI) can be prepared by any convenient method known to those skilled inthe art. For example, U.S. Pat. No. 6,962,930 to Tabakoff et al. andU.S. Pat. No. 7,923,458 to Tabakoff, which are incorporated herein byreference in their entirety, describe the preparation of certainquinoline compounds analogous to those of the present invention, whichreadily can be adapted to the preparation of the aminoquinolinecompounds of Formula (I), (II), (III), (IV) and (V). Scheme 1 provides ageneral scheme for preparing aminoquinoline compounds of Formula (I) andstructurally related or analogous compounds from a 4-amino-substitutedquinoline Compound (A), in which the R substituents are the same asthose in Formula (I). The amino group of Compound (A) is reacted with anactivated acylating Compound (B), comprising a leaving group (LG) thatis reactive toward aromatic amino groups, to form a compound of Formula(I). Substituted quinoline compounds having an amino group in the4-position of the quinoline ring structure, such as Compound (A), havingvarious substitution patterns on the quinoline ring system, and thepreparation thereof, are well known to those of ordinary skill in thechemical arts. Protective groups, such as those disclosed in ProtectiveGroups in Organic Synthesis, 3rd Ed., Green and Wuts, Eds., John Wiley &Sons, Inc. (1999), which is incorporated herein by reference, can beutilized in the preparation of Compound (A), Compound (B) and/or in thecoupling of Compound (A) and Compound (B), as needed or desired tofacilitate the preparation and/or isolation of the compounds of Formula(I).

As used herein, the term “aminoquinoline compound” refers to compoundsas set forth in Formulas (I), (II), (III), (IV), (V) and (VI) asdescribed herein. The aminoquinoline compounds are useful for chronicpain and a variety of other conditions.

The term “alkyl” as used herein is directed to a saturated hydrocarbongroup (designated by the formula C_(n)H_(2n+1)) which isstraight-chained, branched or cyclized (“cycloalkyl”) and which isunsubstituted or substituted, i.e., has had one or more of its hydrogensreplaced by another atom or molecule.

“Aryl” designates either the 6-carbon benzene ring or the condensed6-carbon rings of other aromatic derivatives (see, e.g., Hawley'sCondensed Chemical Dictionary (13 ed.), R. J. Lewis, ed., J. Wiley &Sons, Inc., New York (1997)). Aryl groups include, without limitation,phenyl and naphthyl.

“Heteroaryl” rings are aromatic rings including at least one carbon atomin the ring and one or more, typically from 1-4, atoms forming the ringis an atom other than a carbon atom, i.e., a heteroatom (typically O, Nor S). Heteroaryl includes, without limitation: morpholinyl,piperazinyl, piperidinyl, pyridyl, pyrrolidinyl, pyrimidinyl, triazinyl,furanyl, quinolinyl, isoquinolinyl, thienyl, imidazolyl, thiazolyl,indolyl, pyrrolyl, oxazolyl, benzofuranyl, benzothienyl, benzothiazolyl,benzoxazolyl, isoxazolyl, triazolyl, tetrazolyl, indazolyl, indolinyl,indolyl-4,7-dione, 1,2-dialkyl-indolyl, 1,2-dimethyl-indolyl, and1,2-dialkyl-indolyl-4,7-dione.

“Alkoxy” means —OR where R is alkyl as defined above, e.g., methoxy,ethoxy, propoxy, 2-propoxy and the like.

“Alkenyl” means a linear monovalent hydrocarbon radical of two to sixcarbon atoms or a branched monovalent hydrocarbon radical of three tosix carbon atoms, containing at least one double bond, e.g., ethenyl,propenyl, and the like.

“Alkynyl” means a linear monovalent hydrocarbon radical of two to sixcarbon atoms or a branched divalent hydrocarbon radical of three to sixcarbon atoms, containing at least one triple bond, e.g., ethynyl,propynyl, and the like.

“Halide” and “halo” refer to a halogen atom including fluorine,chlorine, bromine, and iodine.

Substituent groupings, e.g., C₁₋₆ alkyl, are known, and are herebystated, to include each of their individual substituent members, e.g.,C₁ alkyl, C₂ alkyl, C₃ alkyl and C₄ alkyl.

“Substituted” means that one or more hydrogen atoms on the designatedatom is/are replaced with a selection from the indicated group, providedthat the designated atom's normal valency is not exceeded, and that thesubstitution results in a stable compound.

“Unsubstituted” atoms bear all of the hydrogen atoms dictated by theirvalency. When a substituent is, for example, “keto” then two hydrogenson the atom are replaced. Combinations of substituents and/or variablesare permissible only if such combinations result in stable compounds; by“stable compound” or “stable structure” is meant a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

“Pharmaceutically acceptable”, when used in reference to salts orcarriers, refer to materials that are generally accepted as beingsuitable for administration to or contact with the human body orportions thereof. Pharmaceutically acceptable salts are materials inwhich the parent compound (e.g., an aminoquinoline compound of Formula(I) or some other therapeutic agent or excipient is modified by makingacid or base salts thereof. Examples of pharmaceutically-acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines, or alkali or organic salts of acidicresidues such as carboxylic acids. Pharmaceutically acceptable saltsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. Such conventional nontoxic salts includethose derived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Pharmaceuticallyacceptable salts are those forms of compounds, suitable for use incontact with the tissues of human beings and animals without causingexcessive toxicity, irritation, allergic response, or other problems orcomplication, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salt forms of the aminoquinoline compoundsprovided herein are synthesized from the parent compound which containsa basic or acidic moiety by conventional chemical methods. Generally,such salts are prepared, for example, by reacting the free acid or baseforms of these compounds with a stoichiometric amount of the appropriatebase or acid in water or in an organic solvent, or in a mixture of thetwo. Generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17th ed., Mack PublishingCompany, Easton, Pa., 1985, p. 1418, the disclosure of which isincorporated herein by this reference.

“Prodrugs” are considered to be any covalently bonded carriers whichrelease the active parent drug of the aminoquinoline compounds in vivowhen such prodrug is administered to a mammalian subject. Prodrugs ofthe aminoquinoline compounds of the present invention are prepared bymodifying functional groups present in the compounds in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compounds. Prodrugs include compounds whereinhydroxy, amine, or sulfhydryl groups are bonded to any group that, whenadministered to a mammalian subject, cleaves to form a free hydroxyl,amino, or sulfhydryl group, respectively. Examples or prodrugs include,but are not limited to, acetate, formate and benzoate derivatives ofalcohol and amine functional groups in the aminoquinoline compounds ofthe present invention, and the like. Compounds that function effectivelyas prodrugs of the aminoquinoline compounds of the present invention maybe identified using routine techniques known in the art. For examples ofsuch prodrug derivatives, see, for example, (a) Design of Prodrugs,edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol.42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985); (b)A Textbook of Drug Design and Development, edited by Krogsgaard-Larsenand H. Bundgaard, Chapter 5 “Design and Application of Prodrugs,” by H.Bundgaard p. 113-191 (1991); (c) H. Bundgaard, Advanced Drug DeliveryReviews, 8, 1-38 (1992); (d) H. Bundgaard, et al., Journal ofPharmaceutical Sciences, 77:285 (1988); and (e) N. Kakeya, et al., Chem.Pharm. Bull., 32: 692 (1984), each of which is specifically incorporatedherein by reference.

In addition, the invention also includes solvates, metabolites, andpharmaceutically acceptable salts of the aminoquinoline compounds.

The term “solvate” refers to an aggregate of a molecule with one or moresolvent molecules. A “metabolite” is a pharmacologically active productproduced through in vivo metabolism in the body of a specified compoundor salt thereof. Such products may result for example from theoxidation, reduction, hydrolysis, amidation, deamidation,esterification, deesterification, enzymatic cleavage, and the like, ofthe administered compound. Accordingly, the invention includesmetabolites of the aminoquinoline compounds, including compoundsproduced by a process comprising contacting a compound of this inventionwith a mammal for a period of time sufficient to yield a metabolicproduct thereof.

Pharmaceutical Compositions and Treatment Regimens.

In one aspect, the instant invention provides pharmaceuticalcompositions which contain a pharmaceutically effective amount of theaminoquinoline compound in a pharmaceutically acceptable carrier (e.g.,a diluent, complexing agent, additive, excipient, adjuvant and thelike). The aminoquinoline compound can be present for example in a saltform, a micro-crystalline form, a nano-crystalline form, aco-crystalline form, a nanoparticulate form, a mirocparticulate form,and/or an amphiphilic form. The carrier can be an organic or inorganiccarrier that is suitable for external, enteral or parenteralapplications. The aminoquinoline compounds of the present invention canbe compounded, for example, with the usual non-toxic, pharmaceuticallyacceptable carriers for tablets, pellets, capsules, liposomes,suppositories, intranasal sprays, solutions, emulsions, suspensions,aerosols, targeted chemical delivery systems, and any other formsuitable for such use, which are well known in the pharmaceuticalformulation arts. Non-limiting examples of carriers that can be usedinclude water, glucose, lactose, gum acacia, gelatin, mannitol, starchpaste, magnesium trisilicate, talc, corn starch, keratin, colloidalsilica, potato starch, urea and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, liquid or aerosol form.In addition auxiliary, stabilizing, thickening and coloring agents andperfumes can be used.

In particular, the present invention provides pharmaceuticalcompositions useful for treating chronic pain and for prevention ofaddiction relapse, as described herein. The pharmaceutical compositionscomprise at least one aminoquinoline compound as described herein incombination with a pharmaceutically acceptable carrier, vehicle, ordiluent, such as an aqueous buffer at a physiologically acceptable pH(e.g., pH 7 to 8.5), a polymer-based nanoparticle vehicle, a liposome,and the like. The pharmaceutical compositions can be delivered in anysuitable dosage form, such as a liquid, gel, solid, cream, or pastedosage form. In one embodiment, the compositions can be adapted to givesustained release of the aminoquinoline compound.

In some embodiments, the pharmaceutical compositions include, but arenot limited to, those forms suitable for oral, rectal, nasal, topical,(including buccal and sublingual), transdermal, vaginal, parenteral(including intramuscular, intraperitoneal, subcutaneous, andintravenous), spinal (epidural, intrathecal), and central(intracerebroventricular) administration. The compositions can, whereappropriate, be conveniently provided in discrete dosage units. Thepharmaceutical compositions of the invention can be prepared by any ofthe methods well known in the pharmaceutical arts. Some preferred modesof administration include intravenous (iv), topical, subcutaneous, oraland spinal. For systemic administration, the aminoquinoline compoundgenerally will be administered the subject at a dosage in the range ofabout 1 milligram of aminoquinoline compound per kilogram of body mass(mg/kg) to about 200 mg/kg. Typically, the administered dosage should besufficient to provide a concentration of aminoquinoline compound in thesubject of about 1 nanomolar (nM) to about 100 millimolar (mM).

Pharmaceutical formulations suitable for oral administration includecapsules, cachets, or tablets, each containing a predetermined amount ofone or more of the aminoquinoline compounds, as a powder or granules. Inanother embodiment, the oral composition is a solution, a suspension, oran emulsion. Alternatively, the aminoquinoline compounds can be providedas a bolus, electuary, or paste. Tablets and capsules for oraladministration can contain conventional excipients such as bindingagents, fillers, lubricants, disintegrants, colorants, flavoring agents,preservatives, or wetting agents. The tablets can be coated according tomethods well known in the art, if desired. Oral liquid preparationsinclude, for example, aqueous or oily suspensions, solutions, emulsions,syrups, or elixirs. Alternatively, the compositions can be provided as adry product for constitution with water or another suitable vehiclebefore use. Such liquid preparations can contain conventional additivessuch as suspending agents, emulsifying agents, non-aqueous vehicles(which may include edible oils), preservatives, and the like. Theadditives, excipients, and the like typically will be included in thecompositions for oral administration within a range of concentrationssuitable for their intended use or function in the composition, andwhich are well known in the pharmaceutical formulation art. Theaminoquinoline compounds of the present invention will be included inthe compositions within a therapeutically useful and effectiveconcentration range, as determined by routine methods that are wellknown in the medical and pharmaceutical arts.

Pharmaceutical compositions for parenteral, spinal, or centraladministration (e.g. by bolus injection or continuous infusion) orinjection into amniotic fluid can be provided in unit dose form inampoules, pre-filled syringes, small volume infusion, or in multi-dosecontainers, and preferably include an added preservative. Thecompositions for parenteral administration can be suspensions,solutions, or emulsions, and can contain excipients such as suspendingagents, stabilizing agents, and dispersing agents. Alternatively, theaminoquinoline compounds can be provided in powder form, obtained byaseptic isolation of sterile solid or by lyophilization from solution,for constitution with a suitable vehicle, e.g. sterile, pyrogen-freewater, before use. The additives, excipients, and the like typicallywill be included in the compositions for parenteral administrationwithin a range of concentrations suitable for their intended use orfunction in the composition, and which are well known in thepharmaceutical formulation art. The aminoquinoline compounds of thepresent invention will be included in the compositions within atherapeutically useful and effective concentration range, as determinedby routine methods that are well known in the medical and pharmaceuticalarts.

Pharmaceutical compositions for topical administration of theaminoquinoline compounds to the epidermis (mucosal or cutaneoussurfaces) can be formulated as ointments, creams, lotions, gels, or as atransdermal patch. Such transdermal patches can contain penetrationenhancers such as linalool, carvacrol, thymol, citral, menthol,t-anethole, and the like. Ointments and creams can, for example, includean aqueous or oily base with the addition of suitable thickening agents,gelling agents, colorants, and the like. Lotions and creams can includean aqueous or oily base and typically also contain one or moreemulsifying agents, stabilizing agents, dispersing agents, suspendingagents, thickening agents, coloring agents, and the like. Gelspreferably include an aqueous carrier base and include a gelling agentsuch as cross-linked polyacrylic acid polymer, a derivatizedpolysaccharide (e.g., carboxymethyl cellulose), and the like. Theadditives, excipients, and the like typically will be included in thecompositions for topical administration to the epidermis within a rangeof concentrations suitable for their intended use or function in thecomposition, and which are well known in the pharmaceutical formulationart. The aminoquinoline compounds of the present invention will beincluded in the compositions within a therapeutically useful andeffective concentration range, as determined by routine methods that arewell known in the medical and pharmaceutical arts.

Pharmaceutical compositions suitable for topical administration in themouth (e.g., buccal or sublingual administration) include lozengescomprising the aminoquinoline compound in a flavored base, such assucrose, acacia, or tragacanth; pastilles comprising the aminoquinolinecompound in an inert base such as gelatin and glycerin or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier. The pharmaceutical compositions for topicaladministration in the mouth can include penetration enhancing agents, ifdesired. The additives, excipients, and the like typically will beincluded in the compositions of topical oral administration within arange of concentrations suitable for their intended use or function inthe composition, and which are well known in the pharmaceuticalformulation art. The aminoquinoline compounds of the present inventionwill be included in the compositions within a therapeutically useful andeffective concentration range, as determined by routine methods that arewell known in the medical and pharmaceutical arts.

A pharmaceutical composition suitable for rectal administrationcomprises a aminoquinoline compound of the present invention incombination with a solid or semisolid (e.g., cream or paste) carrier orvehicle. For example, such rectal compositions can be provided as unitdose suppositories. Suitable carriers or vehicles include cocoa butterand other materials commonly used in the art. The additives, excipients,and the like typically will be included in the compositions of rectaladministration within a range of concentrations suitable for theirintended use or function in the composition, and which are well known inthe pharmaceutical formulation art. The aminoquinoline compounds of thepresent invention will be included in the compositions within atherapeutically useful and effective concentration range, as determinedby routine methods that are well known in the medical and pharmaceuticalarts.

According to one embodiment, pharmaceutical compositions of the presentinvention suitable for vaginal administration are provided as pessaries,tampons, creams, gels, pastes, foams, or sprays containing a peptide ofthe invention in combination with carriers as are known in the art.Alternatively, compositions suitable for vaginal administration can bedelivered in a liquid or solid dosage form. The additives, excipients,and the like typically will be included in the compositions of vaginaladministration within a range of concentrations suitable for theirintended use or function in the composition, and which are well known inthe pharmaceutical formulation art. The aminoquinoline compounds of thepresent invention will be included in the compositions within atherapeutically useful and effective concentration range, as determinedby routine methods that are well known in the medical and pharmaceuticalarts.

Pharmaceutical compositions suitable for intra-nasal administration arealso encompassed by the present invention. Such intra-nasal compositionscomprise a aminoquinoline compound of the invention in a vehicle andsuitable administration device to deliver a liquid spray, dispersiblepowder, or drops. Drops may be formulated with an aqueous or non-aqueousbase also comprising one or more dispersing agents, solubilizing agents,or suspending agents. Liquid sprays are conveniently delivered from apressurized pack, an insufflator, a nebulizer, or other convenient meansof delivering an aerosol comprising the peptide. Pressurized packscomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas as is well known in the art. Aerosol dosages can becontrolled by providing a valve to deliver a metered amount of thepeptide. Alternatively, pharmaceutical compositions for administrationby inhalation or insufflation can be provided in the form of a drypowder composition, for example, a powder mix of the aminoquinolinecompound and a suitable powder base such as lactose or starch. Suchpowder composition can be provided in unit dosage form, for example, incapsules, cartridges, gelatin packs, or blister packs, from which thepowder can be administered with the aid of an inhalator or insufflator.The additives, excipients, and the like typically will be included inthe compositions of intra-nasal administration within a range ofconcentrations suitable for their intended use or function in thecomposition, and which are well known in the pharmaceutical formulationart. The aminoquinoline compounds of the present invention will beincluded in the compositions within a therapeutically useful andeffective concentration range, as determined by routine methods that arewell known in the medical and pharmaceutical arts.

Optionally, the pharmaceutical compositions of the present invention caninclude one or more other therapeutic agent, e.g., as a combinationtherapy. The additional therapeutic agent will be included in thecompositions within a therapeutically useful and effective concentrationrange, as determined by routine methods that are well known in themedical and pharmaceutical arts. The concentration of any particularadditional therapeutic agent may be in the same range as is typical foruse of that agent as a monotherapy, or the concentration may be lowerthan a typical monotherapy concentration if there is a synergy whencombined with an aminoquinoline compound of the present invention. Forexample, the aminoquinoline compound or a composition comprising thecompound can be administered in conjunction with an anti-cancerchemotherapeutic agent, an antineoplastic agent, and the like (includingmaterials utilized for the treatment and prevention of metastases), fortreating chronic pain associated with cancer therapies. In the case oftreating chronic pain, the amino the aminoquinoline compound or acomposition comprising the compound can advantageously be administeredin conjunction with an analgesic or anti-inflammatory agent (e.g., anopiate, opioid, a non-steroidal anti-inflammatory drug (NSAID), or asteroid), a therapeutic agent for treating a disease (e.g., a diseaseassociated with chronic pain, such as arthritis), and the like, which isdifferent from the aminoquinoline compound.

In another aspect, the present invention provides for the use of theaminoquinoline compounds for treatment of chronic pain. Methods foralleviating chronic pain (e.g., neuropathic pain) comprise administeringto a patient suffering from one of the aforementioned conditions aneffective amount of a aminoquinoline compound. Preferably, theaminoquinoline compound is administered parenterally or enterally. Thedosage of the effective amount of the aminoquinoline compounds can varydepending upon the age and condition of each individual patient to betreated. However, suitable dosages typically range from about 1 mg/kg toabout 200 mg/kg, as discussed above. Such a dose can be administered oneor more times a day, one or more times a week, one or more times permonth, and the like.

As used herein, the terms “reducing”, “inhibiting”, “blocking”,“preventing”, alleviating”, “relieving”, and “antagonist”, whenreferring to a compound (e.g., a peptide), mean that the compound bringsdown the occurrence, severity, size, volume, or associated symptoms of acondition, event, or activity by at least about 7.5%, 10%, 12.5%, 15%,17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 90%, or 100% compared to how the condition, event, oractivity would normally exist without application of the compound or acomposition comprising the compound. The terms “increasing”,“elevating”, “enhancing”, “upregulating”, “improving”, “activating” and“agonist”, when referring to a compound mean that the compound increasesthe occurrence or activity of a condition, event, or activity by atleast about 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%,250%, 300%, 400%, 500%, 750%, or 1000% compared to how the condition,event, or activity would normally exist without application of thecompound or a composition comprising the compound.

EXAMPLES

The following examples are included to demonstrate certain aspects ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples, which representtechniques known to function well in practicing the invention, can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific disclosedembodiments and still obtain a like or similar result without departingfrom the spirit and scope of the invention. The examples are providedfor illustration purposes only and are not intended to be limiting.

Example 1. Preparation of Compounds of Formula (VI)

Derivatives of kynurenic acid containing a tertiary ureido group,including 5,7-dichloro-4-(3,3-diphenylureido)quinoline-2-carboxylic acid(DCUKA, 7a), may be synthesized, as previously described (Snell et al.,2000) through the use of a reactive carbamoyl chloride intermediate(6a-b). However, it is possible to achieve an improvement on thissynthesis due to concomitant ester hydrolysis during the final acylationreaction. One compound embodiment,5,7-dichloro-4-(3,3-dibutylureido)quinoline-2-carboxylic acid (BCUKA,7b), was synthesized via this method in the synthesis phases I-IV asexplained and illustrated in Scheme 2 (Reagents and conditions (I):MeOH, reflux, 16 h. (II): Ph₂O, 250° C., 2 h. (III): (a) ClSO₂NCO, MeCN,reflux, 2 h. (b) HCl, MeOH, RT, 30 min. (IV): NaH, DMF, 0° C. to RT, 16h).

Synthesis Phase I.

3,5-Dichloroaniline (1, 5.00 g, 30.9 mmol) and dimethylacetylenedicarboxylate (2, 3.80 ml, 30.9 mmol) were combined inanhydrous MeOH (60 ml) under nitrogen, and refluxed for 16 hours. Thereaction mixture was cooled to room temperature and evaporated todryness. The resulting yellow solid was recrystallized from MeOH (twice)to give a mixture of cis and trans isomers of the target dimethylanilinomaleate (3) as thin yellow crystals (5.23 g, 17.2 mmol). Theabsorption peak values (in ppm) found in the ¹H NMR spectrum performedin deuterated DMSO were 3.57 & 3.67 (3H, s), 3.72 & 3.80 (3H, s), 5.35 &5.58 (1H, s), 6.98 & 7.12 (2H, Sapp), 7.23 & 7.31 (1H, Sapp), 9.52 &9.64 (1H, br, s).

Synthesis Phase II.

Dimethyl anilinomaleate (3, 3.50 g, 11.5 mmol) was added portion-wise todiphenyl ether (70 ml) at 250° C. The temperature of the resultingsolution was maintained at 250° C. for 2 hours, before being cooled toroom temperature and diluted with hexanes (100 ml). The resultantprecipitate was removed by filtration, washed with hexanes (50 ml), andsuspended in refluxing ethanol, before being filtered to remove solubleimpurities. The solid filtrand was dried under vacuum to give thedesired quinolone carboxylate (4) as an off-white solid (3.10 g, 11.4mmol). The absorption peak values (in ppm) found in the ¹H NMR spectrumperformed in deuterated DMSO were 3.96 (3H, s), 6.59 (1H, s), 7.42 (1H,s), 7.97 (1H, s), 12.05 (1H, br).

Synthesis Phase III.

Chlorosulfonyl isocyanate (1.20 ml, 13.8 mmol) was added to a slurry ofquinoline carboxylate (4, 2.50 g, 9.19 mmol) in anhydrous MeCN (35 ml)at room temperature. The mixture was brought to reflux for 1.5 hours, atwhich point the heating was stopped and a 1.0M solution of HCl inanhydrous MeOH (20 ml) was added. The reaction mixture was allowed tocool to room temperature with stirring until a precipitate formed after1 hour. The precipitate was removed via filtration, washed with MeCN,and air dried. The filter cake was suspended in water (50 ml) to whichsaturated sodium carbonate solution (˜5 ml) was added to pH 10, causingthickening of the suspension. The resultant solid was collected byfiltration, washed with cold water and dried under vacuum (40° C.) togive the target aminoquinoline (5) as an off-white solid (1.82 g, 6.71mmol). The absorption peak values (in ppm) found in the ¹H NMR spectrumperformed in deuterated DMSO were 4.05 (3H, s), 6.04 (2H, s), 7.33 (1H,s), 7.47 (1H, d, J=1.9 Hz), 8.10 (1H, d, J=1.9 Hz).

Synthesis Phase IV.

The acylation of aminoquinoline (5), with concomitant ester hydrolysis,to yield 5,7-dichloro-4-(3,3-dibutylureido)quinoline-2-carboxylic acid(DBCUKA, 7b) was performed as follows; N,N-dibutylcarbamoyl chloride(6b, 96 mg, 0.50 mmol) and aminoquinoline (5, 113 mg, 0.42 mmol) weredissolved in anhydrous DMF (2 ml) and cooled to 0° C. Sodium hydridedispersion in mineral oil (60%, 35 mg, 0.83 mmol) was added and themixture was allowed to warm to room temperature and stirred for 16hours. The reaction was quenched via addition to saturated NH₄Clsolution (1 ml), followed by adjustment to pH 3 with 1.0M aqueous HCl.Extraction with EtOAc (2×10 ml) followed by washing with saturated brine(5 ml) and drying (Na₂SO₄) gave the crude product as a pale yellow oil.Compound purification via silica gel chromatography (9:1 DCM:MeOH) gave5,7-dichloro-4-(3,3-dibutylureido)quinoline-2-carboxylic acid (DBCUKA,7b) as a pale yellow solid (82 mg, 0.20 mmol). The absorption peakvalues (in ppm) found in the ¹H NMR spectrum performed in CDCl₃ were1.00 (6H, t, J=7.4 Hz), 1.36-1.45 (4H, m), 1.64-1.72 (4H, m), 3.39-3.45(4H, m), 5.17 (1H, s), 7.69 (1H, s), 8.30 (1H, s), 9.16 (1H, s).

Carbamoyl chlorides are limited in their commercial availability, and,furthermore, are characterized by high reactivity, especially tohydrolysis, and subsequent poor stability. This is particularly evidentin the case of mono-n-substituted carbamoyl chlorides. Therefore, inorder to prepare mono-n-substituted analogues of kynurenic acid it wasadvantageous to utilize alternative carbamoyl cation equivalents, withattenuated reactivity. Carbamoyl imidazoles (e.g. 9a-d) have been shownto be suitable reactive species for the synthesis of a variety offunctional groups including ureas, thioureas, carbamates, thiocarbamatesand amides. (Grzyb et al., 2005) Derivatives of kynurenic acidcontaining a secondary ureido group were prepared using this approach insynthesis phases V-VII as explained and illustrated in Scheme 3(Reagents and conditions: (V): CDI, DCM, 0° C. to RT, 16 h. (VI): 5,NaH, DMF, 0° C. to RT, 16 h. (VII) TFA, DCM, RT, 16 h).

General Example of Synthesis Phase V.

n-Butylamine (8a, 100 μl, 74 mg, 1.01 mmol) in DCM (1 ml) was added to asolution of CDI (0.197 g, 1.21 mmol) in DCM (5 ml) at 0° C., before thereaction mixture was allowed to warm to RT and stirred overnight. Thesolution was diluted with DCM (10 ml) washed with water (2×10 ml) andbrine (10 ml), dried (Na₂SO₄) and evaporated to dryness to give thetarget carbamoyl imidazole (9a) as a colorless oil (115 mg, 0.69 mmol).The absorption peak values (in ppm) found in the ¹H NMR spectrumperformed in CDCl₃ were 0.97 (3H, t, J=7.4 Hz), 1.42 (2H, qt, J=7.6, 7.3Hz), 1.63 (2H, tt, J=7.3, 7.0 Hz), 3.44 (2H, dt, J=7.0, 6.7 Hz), 6.75(1H, br), 7.07 (1H, s), 7.42 (1H, s), 8.17 (1H, s).

N-(3-(Dimethylamino)propyl)-1H-imidazole-1-carboxamide (9b) was preparedfrom 3-dimethylaminopropylamine (8b) as described in synthesis phase V.The absorption peak values (in ppm) found in the ¹H NMR spectrumperformed in CDCl₃ were 1.77 (2H, tt, J=5.7, 5.5 Hz), 2.32 (6H, s), 2.56(2H, t, J=5.5 Hz), 3.54 (2H, dt, J=5.9, 5.5 Hz), 7.07 (1H, s), 7.27 (1H,s), 8.04 (1H, s), 9.34 (1H, br).

tert-Butyl (3-(1H-imidazole-1-carboxamido)propyl)carbamate (9c) wasprepared from tert-butyl (3-aminopropyl)carbamate (8c) as described insynthesis phase V. The absorption peak values (in ppm) found in the ¹HNMR spectrum performed in CDCl₃ were 1.49 (9H, s), 1.73 (2H, tt, J=5.8,5.7 Hz), 3.30 (2H, dt, J=6.4, 5.7 Hz), 3.48 (2H, dt, J=6.0, 5.8 Hz),4.91 (1H, br), 7.11 (1H, s), 7.52 (1H, s), 7.92 (1H, br), 8.25 (1H, s).

tert-Butyl (3-(1H-imidazole-1-carboxamido)propyl)(methyl)carbamate (9d)was prepared from N-(3-aminopropyl)-N-methyl carbamic acid tert-butylester (8d) as described in synthesis phase V. The absorption peak values(in ppm) found in the ¹H NMR spectrum performed in CDCl₃ were 1.49 (9H,s), 1.74-1.80 (2H, br), 2.87 (3H, s), 3.35-3.43 (4H, m), 7.09 (1H, s),7.53 (1H, s), 8.11 (1H, br), 8.25 (1H, s).

General Example of Synthesis Phase VI.

It was possible to use carbamoyl imidazoles (9a-d) in a manner analogousto carbamoyl chlorides in synthesis phase IV, allowing for a single stepinvolving acylation of the amino quinoline (5) and simultaneous esterhydrolysis. The approach was utilized for the synthesis of4-(3-butylureido)-5,7-dichloroquinoline-2-carboxylic acid (10a) asfollows; N-butyl-1H-imidazole-1-carboxamide (9a, 125 mg, 0.95 mmol) andaminoquinoline (5, 215 mg, 0.79 mmol) were dissolved in anhydrous DMF (4ml) and cooled to 0° C. Sodium hydride dispersion in mineral oil (60%,63 mg, 1.58 mmol) was added and the mixture was allowed to warm to roomtemperature and stirred for 16 hours. The reaction was quenched viaaddition to saturated NH₄Cl solution (3 ml), followed by adjustment topH 3 with 1.0M aqueous HCl. Extraction with EtOAc (2×20 ml) followed bywashing with saturated brine (10 ml) and drying (Na₂SO₄) gave the crudeproduct as a pale orange residue. Compound purification via reversephase (C18) silica gel chromatography (1:1 H₂O:MeCN) gave4-(3-butylureido)-5,7-dichloroquinoline-2-carboxylic acid (10a) as abeige solid (142 mg, 0.39 mmol). The absorption peak values (in ppm)found in the ¹H NMR spectrum performed in deuterated DMSO were 0.92 (3H,t, J=7.3 Hz), 1.31-1.38 (2H, m), 1.44-1.52 (2H, m), 3.16 (2H, dt, J=6.6,6.0 Hz), 7.47 (1H, br), 7.87 (1H, d, J=2.2 Hz), 8.11 (1H, d, J=2.2 Hz),8.68 (1H, s), 9.12 (1H, br).

5,7-Dichloro-4-(3-(3-(dimethylamino)propyl)ureido)quinoline-2-carboxylicacid (10b) was prepared fromN-(3-(dimethylamino)propyl)-1H-imidazole-1-carboxamide (9b) as describedin synthesis phase VI. Due to the zwitterionic nature of the targetcompound, acidification to pH 1/2 was performed with TFA prior toreverse phase (C18) chromatography to afford the product as the TFA saltform. The absorption peak values (in ppm) found in the ¹H NMR spectrumperformed in D₂O were 1.87-2.00 (2H, m), 2.87 (6H, s), 3.12-3.20 (2H,m), 3.21-3.31 (2H, m), 7.13 (1H, s), 7.48 (1H, s), 8.06 (1H, s). Theabsorption peak value (in ppm) found in the ¹⁹F NMR spectrum performedin D₂O was −75.6.

4-(3-(3-((tert-Butoxycarbonyl)amino)propyl)ureido)-5,7-dichloroquinoline-2-carboxylicacid (10c) was prepared from tert-butyl(3-(1H-imidazole-1-carboxamido)propyl)carbamate (9c) as described insynthesis phase VI. The absorption peak values (in ppm) found in the ¹HNMR spectrum performed in deuterated DMSO were 1.39 (9H, s), 1.60 (2H,tt, J=6.8, 6.6 Hz), 2.95-3.02 (2H, m), 3.12-3.18 (2H, m), 6.83 (1H, br),7.45 (1H, br), 7.85 (1H, s), 8.10 (1H, s), 8.65 (1H, s), 9.15 (1H, br).

4-(3-(3-((tert-Butoxycarbonyl)(methyl)amino)propyl)ureido)-5,7-dichloroquinoline-2-carboxylicacid (10e) was prepared from tert-butyl(3-(1H-imidazole-1-carboxamido)propyl)methyl carbamate (9d) as describedin synthesis phase VI. The absorption peak values (in ppm) found in the¹H NMR spectrum performed in deuterated DMSO were 1.39 (9H, s),1.63-1.71 (2H, m), 2.79 (3H, s), 3.08-3.16 (2H, m), 3.19-3.26 (2H, m),7.27 (1H, br), 7.68 (1H, d, J=1.8 Hz), 8.29 (1H, d, J=1.8 Hz), 8.40 (1H,br), 8.97 (1H, br).

General Example of Synthesis Phase VII.

TFA (173 μL, 2.25 mmol) was added to a solution of Boc-protected amine(10c, 103 mg, 0.23 mmol) in DCM (4 ml). After stirring at roomtemperature for 16 hours, the solvent was removed under reduced pressureand the residue was purified directly by reverse phase chromatography(C18, 1:1 H₂O:MeCN) to give the TFA salt form of4-(3-(3-aminopropyl)ureido)-5,7-dichloroquinoline-2-carboxylic acid(10d) as a white solid (64 mg, 0.14 mmol). The absorption peak values(in ppm) found in the ¹H NMR spectrum performed in D₂O (1.0% TFA) were1.61 (2H, tt, J=6.9, 7.1 Hz), 2.72 (2H, t, J=7.1 Hz), 3.04 (2H, t, J=6.9Hz), 7.62 (1H, s), 7.89 (1H, s), 8.71 (1H, s).

5,7-Dichloro-4-(3-(3-(methylamino)propyl)ureido)quinoline-2-carboxylicacid (10f) was prepared from4-(3-(3-((tert-Butoxycarbonyl)(methyl)amino)propyl)ureido)-5,7-dichloroquinoline-2-carboxylicacid (10e) as described in synthesis phase VI. The absorption peakvalues (in ppm) found in the ¹H NMR spectrum performed in D₂O (1.0% TFA)were 1.81 (2H, tt, J=6.8, 7.7 Hz), 2.55 (3H, s), 2.94 (2H, t, J=7.8 Hz),3.22 (2H, t, J=6.8 Hz), 7.78 (1H, d, J=1.9 Hz), 8.01 (1H, d, J=1.9 Hz),8.77 (1H, s). The absorption peak value (in ppm) found in the ¹⁹F NMRspectrum performed in D₂O was −73.4. The compound structures are shownin Scheme 4.

Scheme 4

Compound R 7a (DCUKA)

7b (BCUKA)

10a

10b

10d

10f

Synthesis of 3-(2-butyryl-5,7-dichloroquinolin-4-yl)-1,1-diphenylurea

A.5,7-Dichloro-4-(3,3-diphenylureido)-N-methoxy-N-methylquinoline-2-carboxamide(11). Carbonyldiimidazole (72 mg, 0.44 mmol) and diisoproylethylamine(115 uL, 0.66 mmol) were added to a solution of5,7-dichloro-4-(3,3-diphenylureido)quinoline-2-carboxylic acid (DCUKA;100 mg, 0.22 mmol) in dry N,N-dimethylformamide (15 mL). The reactionmixture was stirred at room temperature under nitrogen for 2 hours,before N,O-dimethylhydroxylamine hydrochloride (86 mg, 0.88 mmol) wasadded. The resulting pale yellow solution was stirred at roomtemperature for a further 16 hours, at which point the solvent wasremoved under reduced pressure and the residue was dissolved in ethylacetate (20 mL) and washed with saturated sodium hydrogen carbonatesolution (2×15 mL) and 0.1M HCl (2×15 mL), followed by water (15 mL) andbrine (10 mL). The organic phase was dried (MgSO₄) and evaporated todryness. The target compound was obtained following purification bychromatography on silica (1:1 Hexanes:EtOAc) as a white solid (81 mg,0.16 mmol, 73%). Rf 0.33 (1:1 Hexanes:EtOAc); M.p. 207-210° C.; 1H NMR(400 MHz, CDCl₃) 3.40 (3H, s), 3.75 (3H, br), 7.28 (1H, s), 7.34-7.37(2H, m), 7.41-7.49 (8H, m), 8.03 (1H, d, J=2.0 Hz), 8.87 (1H, s), 9.39(1H, s).

B. 3-(2-Butyryl-5,7-dichloroquinolin-4-yl)-1,1-diphenylurea (12). An-Propylmagnesium chloride solution in 2-methyltetrahydrofuran (1.0M,1.12 mL, 1.12 mmol) was added dropwise to a solution of5,7-dichloro-4-(3,3-diphenylureido)-N-methoxy-N-methylquinoline-2-carboxamide(11, 70 mg, 0.14 mmol) in dry tetrahydrofuran (10 mL) at −10° C., undernitrogen. Following addition, the reaction mixture was stirred at −10°C. for 30 min, before being allowed to warm to room temperature andstirred for an additional 3 hrs. The reaction was quenched withsaturated ammonium chloride solution (10 mL) and the product, ketone 12,was extracted with ethyl acetate (3×15 mL). The organic extract waswashed with brine (10 mL) and dried (MgSO₄) before being evaporated todryness. The residue was purified via chromatography on silica (4:1Hexanes:EtOAc) to afford the target compound as a pale yellow solid (32mg, 0.07 mmol, 47%). Rf 0.45 (4:1 Hexanes:EtOAc); M.p. 161-164° C.; 1HNMR (400 MHz, CDCl₃) 1.03 (3H, t, J=7.4 Hz), 1.80 (2H, qt, J=7.3, 7.4Hz), 3.24 (2H, t, J=7.3 Hz), 7.28 (1H, s), 7.34-7.38 (2H, m), 7.42-7.49(8H, m), 8.09 (1H, d, J=2.1 Hz), 9.15 (1H, s), 9.31 (1H, s).

Synthesis of5,7-dichloro-4-(3,3-diphenylureido)-N-ethylquinoline-2-carboxamide (13)

Carbonyldiimidazole (143 mg, 0.88 mmol) and diisoproylethylamine (230uL, 1.32 mmol) were added to a solution of5,7-dichloro-4-(3,3-diphenylureido)quinoline-2-carboxylic acid (DCUKA;200 mg, 0.44 mmol) in dry N,N-dimethylformamide (25 mL). The reactionmixture was stirred at room temperature under nitrogen for 2 hours,before ethylamine in THE (2.0M, 0.66 mL, 1.32 mmol) was added. Theresulting pale yellow solution was stirred at room temperature for afurther 16 hours, at which point the reaction was complete. The solventwas removed under reduced pressure and the residue was dissolved inethyl acetate (50 mL) and washed with saturated sodium hydrogencarbonate solution (2×30 mL) and 0.1M HCl (2×30 mL), followed by water(25 mL) and brine (25 mL). The organic phase was dried (MgSO₄) andevaporated to dryness. The target ethylamide 13 was via silica gelchromatography (1:1 Hexanes:EtOAc) as an off-white solid (148 mg, 0.31mmol, 71%). Rf 0.43 (1:1 Hexanes:EtOAc); M.p. 202-205° C.; 1H NMR (400MHz, CDCl₃) 1.31 (3H, t, J=7.2 Hz), 3.56 (2H, q, J=7.2 Hz), 7.28 (1H,s), 7.32-7.37 (2H, m), 7.41-7.49 (8H, m), 7.99 (1H, s), 8.01 (1H, br),9.28 (1H, s), 9.32 (1H, s).

Synthesis of5,7-dichloro-4-(3,3-diphenylureido)-N-isopropylquinoline-2-carboxamide(14)

Carbonyldiimidazole (146 mg, 0.88 mmol) and diisoproylethylamine (230uL, 1.32 mmol) were added to a solution of5,7-dichloro-4-(3,3-diphenylureido)quinoline-2-carboxylic acid (DCUKA;200 mg, 0.44 mmol) in dry N,N-dimethylformamide (25 mL). The reactionmixture was stirred at room temperature under nitrogen for 2 hours,before isopropylamine (110 μL, 1.32 mmol) was added. The resulting paleyellow solution was stirred at room temperature for a further 16 hours,at which point the solvent was removed under reduced pressure and theresidue was dissolved in ethyl acetate (50 mL) and washed with saturatedsodium hydrogen carbonate solution (2×30 mL) and 0.1M HCl (2×30 mL),followed by water (25 mL) and brine (25 mL). The organic phase was dried(MgSO₄) and evaporated to dryness. The target isopropylamide 14 was viasilica gel chromatography (1:1 Hexanes:EtOAc) as a white solid (182 mg,0.37 mmol, 84%). Rf 0.52 (1:1 Hexanes:EtOAc); M.p. 197-199° C.; 1H NMR(400 MHz, DMSO-d6) 1.23 (6H, d, J=6.8 Hz), 4.15 (1H, m), 7.34-7.39 (2H,m), 7.46-7.55 (8H, m), 7.76 (1H, d, J=1.9 Hz), 8.11 (1H, d, J=1.9 Hz),8.55 (1H, d, J=8.2 Hz), 9.00 (1H, s), 9.21 (1H, s).

Example 2. Effect of DCUK Compounds on Ligand Binding to Delta OpiateReceptor, Cannabinoid CB1 Receptor and GABA-A Receptor and OtherNeurotransmitter Receptors, Ion Channels and Transporters

This example compares the effect of DCUKA, BCUKA, or DCUK-OEt on thebinding of ligands defining the delta opiate receptor, the cannabinoidCB1 receptor, and the agonist and benzodiazepine sites on theGABA-receptor, as well as other receptors and neurotransmittertransporters, performed using membrane preparations from cultured cellsor from rat brain.

These assays were performed by the Psychoactive Drug Screening Program(PDSP) (NIMH). Detailed methods can be found at the PDSP websitePDSP(dot)med(dot)unc(dot)edu/pdspw/binding(dot)php. Table 1 is takenfrom the PDSP website, as well as the lists the cell lines used forbinding assays in Tables A, B, and C. The following brief description,including Tables 1, A, B, and C, is quoted from the website.

“To make membrane fractions from stably transfected cell lines, cellsare subcultured in 15-cm dishes and grown to 90% confluency. The nextday, the cells are rinsed with PBS, scraped off into 50 ml conicaltubes, and pelleted by centrifugation (1000×g, 10 min at 4° C.). Thecell pellet is resuspended in chilled (4° C.) lysis buffer (50 mM TrisHCl buffer, pH 7.4) and triturated gently for hypotonic lysis. Thesuspension is then centrifuged at 21,000×g for 20 min at 4° C. to obtaina crude membrane fraction pellet. The fresh membrane pellet is thenresuspended in 3 volumes of cold lysis buffer, and is immediatelysubjected to the Bradford protein assay to determine proteinconcentration, followed by a saturation binding assay (see followingsection for detail) to determine receptor expression level and theaffinity of a selected radioligand (K_(d)). Based on the receptorexpression level and the K_(d) value, the fresh membrane suspension isstored at −80° C. freezer in small aliquots, such that one aliquot issufficient for one 96-well plate to have at least 500 cpm/well whenassayed at 0.5-1.0×K_(d) value of the appropriate radioligand.”

TABLE 1 List of cell lines and targets used by the PDSP to make membranepellets for binding assays. All clones are stable lines, whereastransiently transfected cells are marked with “*”. Clones are humanunless noted. From PDSP website. Receptor Note Parental cells Media (seedetail below the table) Serotonin (5HT) 5-HT1A stable CHO 500 G4185-HT1B stable HEK 500 G418 5-HT1D * HEKT COS/HEK 5-HT1E stable HEK 500G418 5-HT2A (rat) stable 3T3 500 G418 5-HT2A * HEKT COS/HEK 5-HT2Bstable HEK 2 μg/ml Puromycin 5-HT2C Flp-IN HEK DMEM 100 μg/ml HygromycinB 5-HT3 * HEKT COS/HEK 5-HT5A Flp-In CHO DMEM/F-12 200 μg/ml HygromycinB 5-HT6 stable HEK 500 G418 5-HT7A stable HEK 2 μg/ml Puromycin DopamineD1 * HEKT COS/HEK D2 stable fibroblast COS/HEK D2L stable CHO F-12/10%FBS 400 G418 D3 (rat) * HEKT COS/HEK D3 * HEKT COS/HEK D4 stableDMEM/F12 10% CS Fe+ D5 * HEKT COS/HEK Opioid Mu, MOR stable HEK 200 G418Delta, DOR stable HEK 200 G418 Kappa, KOR (rat) stable HEK 500 G418Kappa, KOR stable HEK 500 G418 Nociceptin, NOP * HEKT COS/HEKNeurotransmitter Transporters SERT stable HEK 500 G418 NET stable HEKhNET (250 G418) DAT stable HEK hDAT (350 G418) Vasopressin and OxytocinV1A stable CHO V1A & OT media V2 stable CHO V2 & V1B media V1B stableCHO V2 & V1B media OT stable CHO V1A & OT media Prostaglandin EP-3 *HEKT COS/HEK EP-4 * HEKT COS/HEK Adrenergic alpha 1A stable 500 G418alpha 1B * HEKT alpha 1D stable 500 G418 alpha 2A stable MDCK 500 G418alpha 2B * HEKT COS/HEK alpha 2C stable MDCK 500 G418 beta 1 CHO Flp-InDMEM/F12 200 μg/ml Hygromycin B beta 2 HEK Flp-In DMEM 100 μg/mlHygromycin B beta 3 HEK Flp-In DMEM 100 μg/ml Hygromycin B Muscarinicacetylcholine M1 stable CHO 500 G418 M2 stable CHO 500 G418 M3 stableCHO 500 G418 M3D CHO Flp-In DMEM/F12 100 μg/ml Hygromycin B M4 stableCHO 10% FBS F12 M5 stable CHO 500 G418 Nicotinic acetylcholine α2β3 HEK500 G418 α2β4 HEK 500 G418 α3β2 HEK 500 G418 α3β4 HEK 500 G418 α4β2 HEK500 G418 α4β4 HEK 500 G418 α7 HEK 500 G418 Histamine H1 stable HEK 500G418 H2 (in progress) stable HEK 500 G418 H3 HEK Flp-In DMEM 100 μg/mlHygromycin B H4 (in progress) 500 G418 Cannabinoid CB1 (in progress) HEK500 G418 CB1 HEK Flp-In DMEM 100 μg/ml Hygromycin B CB2 HEK Flp-In DMEM100 μg/ml Hygromycin B Adenosine A1 * HEKT COS/HEK A2A * HEKT COS/HEKA2A HEK 500 G418 A2B * HEKT COS/HEK A3 * HEKT COS/HEK MelanocortinMC-1 * HEKT COS/HEK MC-2 * HEKT COS/HEK MC-3 * HEKT COS/HEK MC-4 * HEKTCOS/HEK MC-5 * HEKT COS/HEK Purinergic P2Y P2Y1 Astrocyte line 500 G418P2Y2 Astrocyte line 500 G418 P2Y4 Astrocyte line 500 G418 P2Y6 Astrocyteline 500 G418 P2Y11 Astrocyte line 500 G418 P2Y12 Astrocyte line 500G418 Trace Amine TA-1 * HEKT COS/HEK TA-3 * HEKT COS/HEK TA-4 * HEKTCOS/HEK TA-5 * HEKT COS/HEK Parental cells Media (see detail below (seedetail below Receptor Note the table) Media the table) Lysophospholide(LPA) LPA1 * HEKT COS/HEK LPA2 * HEKT COS/HEK LPA3 * HEKT COS/HEKTachykinin (NK) NK1 HEK 500 G418 NK2 HEK 500 G418 NK3 HEK 500 G418mGluRs mGluR1 (in progress) * HEKT 500 G418 mGluR2 (in progress) * HEKT500 G418 mGluR3 (in progress) * HEKT 500 G418 mGluR4 (in progress) *HEKT 500 G418 mGluR5 CHO 2 μg/ml Puromycin mGluR5 (in progress) * HEKT500 G418 mGluR6 (in progress) * HEKT 500 G418 mGluR7 (in progress) *HEKT 500 G418 mGluR8 (in progress) * HEKT 500 G418 Others Ghrelin HEKFlp-In DMEM 100 μg/ml Hygromycin B PAR1 Lung Fibroblast, 500 G418 PAR1KO SMO * HEKT COS/HEK SMO (in progress) HEK 500 G418 CCK2 CHO 500 G418Orexin-2 * HEKT COS/HEK GPR58 * HEKT COS/HEK GPR61 * HEKT COS/HEKGPR62 * HEKT COS/HEK GPR40 * HEKT COS/HEK GPR41 * HEKT COS/HEK GPR43 *HEKT COS/HEK Il-1 imidazoline * HEKT COS/HEK HERG-K⁺ Channel HEK 500ug/ml G418 PC12 COS/HEK

“Radioligands and their concentrations, reference compounds, and buffersfor radioligand binding assays are listed in Tables A, B, and C. Theconcentrations of radioligand used for competition binding assay areusually at or near the K_(d) value or as listed. Historical referenceK_(i) values from the last 6 months are also included.”

TABLE A Opioid Receptors. From PDSP website. K_(d) for [³H] Compoundbinding in nM Reference Target Radioligand (N) References K_(i) (nM) DOR[³H]DADLE 1.85 ± 0.15 (2) Naltrindole  0.81 ± 0.08 KOR [³H]U69593 1.07 ±0.10 (21) Salvinorin A  1.93 ± 0.45 MOR [³H]DAMGO 1.73 ± 0.14 (6) DAMGO, 2.62 ± 0.22 Morphine NOP [³H]N/OFQ 0.74 ± 0.22 (4) JDTiC B612111 12.05± 1.47  6.58 ± 1.42 Standard binding buffer: 50 mM Tris HCl, 10 mMMgCl2, 0.1 mM EDTA, pH 7.4, RT. Standard wash buffer: 50 mM Tris HCl, pH7.4, 4° C. to 8° C.

TABLE B Cannabinoid Receptors from PDSP website K_(d) for [³H] CompoundReference binding in nM K_(i) Target Radioligand (N) References (nM) CB1[³H]CP55940 1 mM WIN55212-3 18.5 ± 3.0  (rat brain) CB2 [³H]CP55940 2 nMWIN55212-3 9.4 ± 2.6 CP55940 4.95 ± 0.55 Cannabinoid Binding Buffer: 50mM Tris HCl, 5 mM MgCl₂, 1 mM EDTA, 1 mg/ml BSA, pH 7.4, RT CannabinoidWash Buffer: cannabinoid binding buffer + 1 mg/ml BSA, pH 7.4, cold

TABLE C GABA receptors From PDSP website K_(d) for [³H] Compound bindingReference in nM K_(i) Target Radioligand (N) References (nM) GABA/PBR[³H]PK11195 1 PK11195 27.6 ± 2.3  (rat brain) Ro5-4864 GABAA[³H]Muscimol 1.075.0 GABA 241 ± 26  (rat brain) GABAA/BZP[³H]Flunitrazepam 0.5 Diazepam 1.50 ± 0.08 (rat brain) ClonazepamGABA/PBR binding buffer: 50 mM Tris Acetate, pH 7.4, RT. Benzodiazepine(BZP) binding buffer: 50 mM Tris HCl, 2.5 mM CaCl₂, pH 7.4, RT. Standardwash buffer: 50 mM Tris HCl, pH 7.4, cold

“Saturation binding assays are usually performed immediately after themembrane fraction is obtained and protein concentration is determined(see above section for membrane preparations) to measure receptorexpression level (B_(max)) and binding affinity (K_(d)) of a selectedradioligand. Saturation binding assays are carried out in 96-well platesin a final volume of 125 μl per well. In brief, 25 μl of radioligand isadded to each well of a 96-well plate, followed by addition of 25 μlbinding buffer (for total binding) or 25 μl reference compound at finalconcentration of 10 μM (for nonspecific binding). The reaction startsupon addition of 75 μl of fresh membrane protein (typically 25 to 50 μgper well) and the reaction is usually incubated in the dark at roomtemperature for 90 min. The reaction is stopped by vacuum filtrationonto cold 0.3% polyethyleneimine (PEI) soaked 96-well filter mats usinga 96-well FILTERMATE harvester, followed by three washes with cold washbuffers . . . Scintillation cocktail is then melted onto themicrowave-dried filters on a hot plate and radioactivity is counted in aMICROBETA counter. IC₅₀ values are calculated and converted to K_(i)values by standard methods (Cheng and Prusoff, 1973).”

Results obtained for effects of compounds derived from Formula (VI) atcannabinoid (CB1) receptors, delta opiate receptors and GABA-A receptorsare shown in Table 2.

TABLE 2 Radioligand Displacement Studies: Binding Constants (K_(i) inμM) for DCUKA, BCUKA, and DCUK-OEt Delta GABAsite, BDZsite, Opiate CB1GABA-A GABA-A Compound Receptor Receptor Receptor Receptor DCUKA 4.5 ±0.73 11.0 ± 2.7  6.6 ± 1.8 >10 BCUKA 1.7 ± 0.41 N.D. N.D. 0.63 ± 0.04DCUK-OEt >10 7.4 ± 2.4 1.7 ± 0.3 N.D. N.D. = not determined

Table 3 lists receptors, ion channels and transporters that were foundto have low or no affinity (Ki>10 μM) for DCUKA, BCUKA, and DCUK-OEt.

TABLE 3 Lack of Affinity (K_(i) > 10 μM) for DCUKA and BCUKA andDCUK-OEt across 26 Receptor/Transporter/Channel Proteins Serotonin RsDopamine Rs 5-HT1A D1 5-HT1B D2 5-HT2A Opiate Rs 5-HT2B μ 5-HT3 κAdrenergic Rs (alpha adrenergic) Prostaglandin Rs α2β2 EP2 α2β4Muscarinic Cholinergic Rs α3β2 M1 α3β4 M2 α4β2 Metabotropic Glutamate Rsα4β2 mGluR5 Adrenergic Rs (beta adrenergic) Ionotropic glutamate Rs β1NMDA channel binding site β2 Kainate Voltage sensitive Ca** channel(L-type) Serotonin Transporter CaV1.2 Vasopressin V1A

Example 3: Effect of DCUKA, BCUKA and DCUK-OEt on Delta Opiate Receptoror Cannabinoid CB1 Receptor Function

This example illustrates the effect of DCUK compounds on cyclic AMPproduction in cells (HeLA or HEK293) transfected with the cannabinoidCB1 receptor or the delta opiate receptor, dopamine D₁ receptor, andType 5 adenylyl cyclase or Type 7 adenylyl cyclase.

HeLa cells and HEK293 cells were obtained from American Type CultureCollection (Manassas, Va.). Cells were cultured in flasks (225 cm²)containing 39 ml of MEM containing 10% fetal bovine serum, penicillin(50 μg/ml), streptomycin (50 μl), and neomycin (100 μg/ml). The flaskswere maintained in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. Transfection was performed by the method of DNA precipitation withcalcium phosphate (Chen and Okayama, 1987) 1 day after HeLa cells orHEK293 cells were transferred to small flasks (75 cm²) at the density ofapproximately 60 to 80% confluence. Plasmid DNA containing adenylylcyclase (AC) cDNA (11.5 μg), plasmid DNA carrying the human D_(1A)dopamine receptor cDNA or any other receptor plasmid used in this study(3 μg), and pCMS-EGFP (1 μg) were used for each small flask (75 cm²)containing 13 ml of the culture medium. The amount of DNA per flask wasadjusted to a total of 26 μg using vector DNA. Transfection efficiencywas routinely monitored by observing the expression of enhanced greenfluorescent protein by a epifluorescence microscope equipped with afilter set (excitation, 480/40 nm; emission, 535/50 nm) and a dichroicmirror (T89002bs; Chroma Technology, Rockingham, Vt.). Aftertransfection, cells were harvested and transferred into 24-well cultureplates. The transfected cells were cultured for 1 to 2 days beforecyclic AMP (cAMP) accumulation experiments were carried out.

The drugs used for the pharmacological treatment were prepared as stocksolution. DCUK-OEt, DCUKA and BCUKA were dissolved in dimethyl sulfoxide(DSMO, 10 mM). D_(1A) receptor agonist, dopamine (DA, Sigma) wasdissolved in 0.9% N_(a2)S₂O₅, 1 mM HCl (10 mM); delta opiate receptor(DOR) agonist, DPDPE (Sigma) was dissolved in 1 M acetic acid (1 mM);DOR antagonist Naltrindole (Sigma) was dissolved in H₂O (10 mM).

The activity of AC in the transfected cells was assessed by the cAMPaccumulation assay as described previously (Kou and Yoshimura, 2007).Briefly, after labeling the intracellular ATP pool with 3.0 μCi/ml of[2,8-³H]adenine, cells were incubated in DMEM (0.5 ml/well) withoutphenol red, buffered to pH 7.1 with 20 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) for 30minutes at 37° C.

To determine the effect of the DCUK-OEt on CB₁R or DCUKA and BCUKA onDOR, the cells were treated with the phosphodiesterase inhibitor, IBMX(500 μM) for 10 minutes at 37° C., followed by the addition of D_(1A)receptor agonist, DA (1 μM or 10 μM), in the presence of the appropriatereceptor agonist or agonist plus antagonist for 5 minutes. The followingagonists and antagonists are used: DCUKA (0.25-20 μM); DCUK-OEt (10 μM);BCUKA (5, 10, 20 μM); CB1 receptor agonist, ACEA (10 μM); CB1 receptorantagonist, AM251 (10 μM); DOR agonist, DPDPE (1 μM); DOR antagonistNaltrindole (10 μM).

The reaction was terminated by adding 50 μl of 100% (w/v)trichloroacetic acid. Cells were exposed to the same amount (10 μl) ofDMSO regardless of the DCUKA analog employed.

This amount of DMSO did not have any significant effect on the cAMPaccumulation (Kou and Yoshimura, 2007). ATP and cAMP contents of eachwell were separated through DOWEX 50 and neutral alumina columns asdescribed previously (Salomon et al., 1974) and quantified by liquidscintillation spectrometry. [α-³²P]ATP and [8-¹⁴C]cAMP were added asinternal standards to monitor the recovery of ATP and cAMP throughcolumn chromatography. Conversion of [³H] ATP into [³H]cAMP wascalculated as follows: [³H]cAMP (%)=[³H]cAMP (cpm)/([³H]ATP(cpm)+[³H]cAMP (cpm))−100. To obtain cAMP accumulation during the1-minute DA-stimulation period, [³H]cAMP (%) values obtained from cellswhich underwent only the 10-minute 1BMX±DCUKA incubation period beforethe addition of trichloroacetic acid, were subtracted from the valuesobtained from cells which underwent the 1-minute DA-stimulation period.The effect of DCUK-OEt, DCUKA or BCUKA was calculated as the percentchange of cAMP accumulation in the presence of DCUK-OEt or BCUKA overthat in its absence. The experiments were carried out in triplicate.

In these experiments, the agonist activity of DCUKA, BCUKA or DCUK-OEtat cannabinoid CB1 and delta opiate receptors (DOR) is determined bymeasuring the effect of DCUKA, BCUKA or DCUK-OEt, in the presence ofdopamine (acting at the D1 receptor), on modulation of adenylyl cyclase(AC) activity, which produces changes in cyclic AMP (cAMP) levels.Dopamine stimulates the activity of Type 5 and Type 7 adenylyl cyclases(AC5, AC7). Under conditions of DA-stimulated AC activity, activation ofDOR or CB1 receptors by agonists leads to increased activity of AC7(increased cAMP), but inhibition of activity of AC5 (decreased cAMP).

For the experiments with BCUKA and DCUK-OEt (FIGS. 1 and 2), the valueof cAMP accumulation was compared among all treatment groups by one-wayANOVA followed by the Student-Newman-Keuls pairwise comparison method.Significance level was determined as p<0.05.

FIG. 1 demonstrates the effect on cAMP accumulation of the CB1 receptoragonist ACEA and DCUK-OEt in HeLa cells transfected with the AC7isoform. One way ANOVA revealed an overall significant effect on cAMPaccumulation (F (5,18)=3.999, P=0.013). ACEA increased the accumulationof cAMP (P<0.001), and this effect was reversed by AM251, the antagonistof the CB1 receptor (P=0.065 vs DA+ACEA group and P=0.75 vs DA group).Like the CB1 receptor agonist, DCUK-OEt produced a significant increasein cAMP accumulation (P=0.05) in cells which were activated by DA, andthe CB1 receptor antagonist AM251 eliminated the DCUK-OEt effect.However, when ACEA and DCUK-OEt were added together, the accumulation ofcAMP was noted to be less than in the presence of ACEA above. Thisresult illustrates that DCUK-OEt acts as weak partial agonist at the CB1cannabinoid receptor.

FIG. 2 illustrates the effect on cAMP accumulation of BCUKA in HEK293cells transfected with the DA receptor, the delta opiate receptor, andthe AC7 isoform. One way ANOVA revealed an overall significant effect oncAMP accumulation (F (6,14)=25.94, P<0.001). BCUKA (10 and 20 μM), inthe presence of dopamine, increased cyclic AMP accumulation (P=0.05),and this effect was reversed by the delta opiate receptor antagonist,naltrindole. In addition to the data in the figure, cAMP accumulation inthe presence of DA was significantly increased by DPDPE (P<0.001) andthis effect was reversed by naltrindole. Taken together, these resultsillustrate that BCUKA acts as an agonist at the delta opiate receptor.

FIG. 3 illustrates the concentration related inhibitory effect on cAMPaccumulation by DCUKA in HeLa cells transfected with delta opiatereceptor and the AC5 isoform. Regression analysis determined that theIC₅₀ for DCUKA was 3.90±0.47 μM. The effect of DCUKA was reversed by thedelta opiate receptor antagonist Naltrindole (not shown). This resultillustrates that DCUKA acts as an agonist at the delta opiate receptor.

Example 4: Effect of DCUKA and DCUK-OEt on GABA-A Receptor Function

This example illustrates the effect of DCUKA, DCUK-OEt, DCUK-butanone,DCUK-ethylamide, and DCUK-isopropyl amide on chloride fluxes resultingfrom activation of GABA-A receptors expressed in Xenopus oocytes.

Xenopus laevis oocytes were injected with mRNA encoding various GABA-Asubunits. Responses to GABA and modulators were recorded 3-5 days later,using two-electrode voltage clamp (Borghese et al., 2014). Xenopuslaevis frogs were obtained from Nasco (Fort Atkinson, Wis., USA). Thecomplementary DNAs encoding the GABA_(A) subunits rat α1 and γ2s andhuman β2 were provided by Dr. M. H. Akabas and Dr. Paul Whiting,respectively. The in vitro transcription of α1, β2, and γ2s subunits wasperformed using mMessage mMachine® (Life Technologies, Grand Island,N.Y.). After isolation of Xenopus laevis oocytes, they were injectedwith capped complementary RNAs encoding α1, β2, and γ2s subunits in aratio 2:2:20 ng. The injected oocytes were incubated at 15° C. insterilized Barth's solution [MBS; composition: 88 mM NaCl, 1 mM KCl, 10mM HEPES, 0.82 mM MgSO₄, 2.4 mM NaHCO₃, 0.91 mM CaCl₂, and 0.33 mMCa(NO₃)₂, pH 7.5] for 3-5 days before recording. All surgery wasperformed according to an approved institutional protocol. The responsesof GABA_(A)Rs expressed in oocytes were studied through two-electrodevoltage clamp (Oocyte Clamp OC-725C, Warner Instruments, Hamden, Conn.),recording through a POWERLAB 4/30 system (ADInstruments, ColoradoSprings, Colo.). The oocyte was placed in a chamber perfused with MBS,and voltage-clamped at −70 mV. GABA applications lasted for 20-30 s andthe interval between them was 5-15 min. In order to test modulation, theagents were first pre-applied for 1 min and then co-applied with GABA.The application sequence was as follows: Maximal GABA, EC₁₀ GABA, EC₁₀GABA, pre-application of the drug immediately followed by aco-application with EC₁₀ GABA, EC₁₀ GABA.

FIG. 4A compares the effects of DCUKA and DCUK-OEt alone or in thepresence of GABA on chloride currents generated by activation of GABA-Areceptors. DCUKA and DCUK-OEt had no effect on chloride currents bythemselves, but significantly enhanced GABA-induced currents. Theresults indicate that DCUKA and DCUK-OEt are positive allostericmodulators (PAM) of the actions of GABA.

FIG. 4B demonstrates that the DCUK compounds are not acting by bindingto the benzodiazepine site on the GABA-A receptor since the specificbenzodiazepine antagonist had no significant inhibitory effect on thepotentiation of the GABA response either by DCUK-OEt or DCUKA.

FIG. 4C illustrates the structure/activity relationship on the efficacyof five DCUK derivatives. This data indicates that DCUK-OEt andDCUK-butanone were most efficacious and suggests that an ester or amidesubstituent at the 2-position of DCUK is important for the positiveallosteric properties of the DCUK compounds.

FIG. 4D provides a graphic representation of the effects of DCUK-OEt onGABA-A receptors having different subunit compositions which areimportant in the magnitude of the positive allosteric modulationachieved by DCUK-OEt. The tested subunit combinations are listedstarting with the most responsive to DCUK-OEt. This rank order, as shownin FIG. 4D does not mirror any currently known positive allostericmodulator of the GABA-A receptor.

Example 5. Effect of DCUKA, BCUKA and DCUK-OEt on Reversal ofNeuropathic Pain

This example illustrates the ability of DCUKA, BCUKA and DCUK-OEt toreverse neuropathic pain, measured as mechanical or thermal pain,induced by cisplatin (cancer chemotherapy), Complete Freund's Adjuvant(CFA) (inflammatory pain), or diabetes (streptozotocin-induced pain).

All studies were performed in accordance with the NIH Guide for the Careand Use of Laboratory Animals.

Drugs.

For in vivo studies, DCUKA or BCUKA or DCUK-OEt, or gabapentin, wereprepared in a 50% gelatin/50% canola oil emulsion (this emulsion wasused as vehicle). The gelatin was prepared by adding 0.8 g of gelatin(Knox, Kraft Foods North America, Tarrytown, N.Y.) and 0.06 g tartaricacid (McCormick and Co., Inc., Hunt Valley, Md.) to 30 ml of purifiedwater. The solution was heated at 98° C. for 20 minutes, then cooled to50° C. Six ml of 95% alcohol and water were added to make 50 ml ofgelatin. Various amounts of DCUKA or BCUKA or DCUK-OEt, or gabapentin,were added to 5 ml of canola oil (Safeway Inc., Pleasanton, Calif.) withstirring and sonication (VWR BIOSONIK IV, 70%) for 5 minutes, and thedrug suspensions were then added to 5 ml of gelatin with stirring andsonication. The emulsions were diluted with vehicle as needed and warmedto 37° C. for oral administration to animals. Immediately prior to oralgavage, the emulsion was stirred using a vortex mixer.

Three different agents were used to produce neuropathic pain. Cisplatin(Sigma-Aldrich, St. Louis, Mo.) was dissolved in 0.9% saline solution.Streptozotocin (Sigma-Aldrich) was dissolved in 20 mM sodium citratebuffer, pH=4.5. Complete Freund's Adjuvant (CFA) was obtained fromSigma-Aldrich.

Measurement of Mechanical Hyperalgesia.

These studies were performed on male Sprague-Dawley rats (Taconic,Germantown Pa.) at approximately eight weeks of age. Rats were housed inan AAALAC-accredited facility with regulated lighting, temperature andhumidity. Pain was tested using an electronic von Frey anesthesiometer(IITC Life Science), Woodland Hills, Calif.). Rats were placed insuspended chambers with a metal mesh floor and were allowed to acclimatefor approximately 20 minutes. Mechanical stimuli were applied to themid-plantar surface of the hind paw(s). Two different methods were used.In the first, a set of von Frey filaments with different strength ranges(g) was used, and filaments of increasing strength were applied to thepaw. The force generated with each filament application was displayed onthe electronic sensor. Each filament was applied five times, until a pawwithdrawal response occurred (“flinch” after filament application). Whena filament produced paw withdrawal in four out of five tests, or themaximal stimulus (10% of body weight) was reached, testing was stopped.The average of the four values was used to calculate the paw withdrawalthreshold in g. In the second method, a semi-flexible filament wasplaced against the mid plantar surface of the paw with escalatingpressure, until paw withdrawal was observed. The pressure (force in g)at paw withdrawal (paw withdrawal threshold) was recorded by anelectronic transducer. Five measurements were taken per test, and theaverage value was calculated. In both methods, to avoid sensitization, athree-minute interval was imposed between measurements.

Acute Effects of DCUKA, BCUKA, DCUK-OEt and Gabapentin to ReverseNeuropathic Pain (Mechanical Hyperalgesia) Caused by Cisplatin, CompleteFreund's Adjuvant (CFA) or Streptozotocin (STZ).

Acute Effect of DCUKA on Cisplatin-induced Pain:

Two methods were used for cisplatin administration. 1) Cisplatin wasdissolved in 0.9% saline (1 mg/ml) and injected into the tail vein in avolume of 1.5 or 2.5 ml/kg of body weight. The intravenous injection ofcisplatin was followed by an injection of the same amount of saline(Joseph and Levine, 2009). The cisplatin doses were 1.5 or 2.5 mg/kg inindividual experiments. 2) Cisplatin was dissolved in 0.9% saline andadministered by intraperitoneal injection on days 1, 4, 8 and 12. Thecisplatin doses were 2 mg/kg, 1 mg/kg, 2 mg/kg and 2 mg/kg,respectively, for a total dose of 7 mg/kg. A fresh solution of cisplatinwas prepared every day before injection, and 0.9% saline (2 ml) wasinjected subcutaneously after the cisplatin injection (to avoidnephrotoxicity). In all experiments, rats were tested for baseline painsensitivity (mechanical pain threshold) prior to any cisplatintreatment. The experimental designs for the studies using intravenousinjections were as follows: (1) Starting at one hour after cisplatininjection, rats were given vehicle (canola oil/gelatin) orally (byintragastric gavage) twice daily (every 12 hours) for three days (theserats are controls for the study on prevention of cisplatin-induced pain,see below). On the fourth day, rats were again tested for mechanicalpain threshold. On the fifth day, rats were given 50 mg/kg DCUKA orvehicle and mechanical pain threshold was tested one hour later. 2) Onday 4 after cisplatin treatment, rats were given various doses of DCUKA(12.5, 25, 50, or 75 mg/kg), or vehicle, by intragastric gavage, andmechanical pain threshold was tested one hour later. 3) On day 6 aftercisplatin treatment, the mechanical pain threshold was measured, andrats were given 50 mg/kg DCUKA, or vehicle, orally. The mechanical painthreshold was tested one hour later. 4) On day 6 after cisplatintreatment, rats were given various doses of DCUKA (25, 50 or 75 mg/kg)or vehicle, and one hour later, mechanical pain threshold was measured.The experimental design for experiments in which cisplatin wasadministered intraperitoneally were as follows: on day 14, following thecisplatin treatments (days 1, 4, 8, 12), mechanical pain threshold wastested. Rats were then given 50 mg/kg DCUKA, or vehicle, orally and themechanical pain threshold was measured one hour later. Data are reportedas the ratio of the mechanical pain threshold measured after DCUKAtreatment to the baseline pre-cisplatin mechanical pain threshold(measured on the same paw).

Data Analysis of Cisplatin Experiments: Acute Effect of DCUKA andMeta-Analysis: Depending on the experimental design, analysis consistedof either 1-way ANOVA with repeated measures or 2-way ANOVA withrepeated measures (Proc Mixed, SAS v9.3, Cary, N.C.). Treatment and timeare the main fixed independent effects tested. In one experimenttreatment, time and the interaction between the two were assessed. Theanimal identification number was used as a repeated measure as there aremultiple measurements on one animal, including pre and post treatmentand left and right paw. All models were tested for equal variances amongtreatment groups (Barlett's test for homogeneity of variances) andnormality (Kolmogorov-Smirnov goodness of fit test). If the data did notpass these assumptions, we adjusted accordingly in the mixed model. Someanalyses used Fisher's LSD post hoc tests to compare statisticalsignificance (p-value<0.05) between the different treatment doses andall analyses used Fisher's LSD post hoc tests to compare statisticalsignificance between treatment group and the baseline value.

Experiments were included in the meta-analysis (van Houwelingen et al.,2001) if they met the following requirements: 1. Mechanical pain wasmeasured using a von Frey test, 2. Cisplatin treatment successfullyinduced pain (25% decrease of mechanical pain threshold) and 3.Mechanical pain was measured within 90 minutes after DCUKAadministration. A mixed-model using DCUKA dose as a fixed independentvariable with 5 different class levels, study identification as both arandom and a repeated measure and rat identification as a random effectwas used to determine the overall effectiveness of DCUKA on neuropathicpain (Proc Mixed, SAS v9.3, Cory, N.C.). Fisher's LSD post-hoc testswere used for pairwise comparisons of the DCUKA doses.

Comparison of the Effects of DCUKA, BCUKA and Gabapentin onCisplatin-Induced Pain. Cisplatin was administered intraperitoneally ondays 1 (2 mg/kg), 4 (1 mg/kg), 8 (2 mg/kg) and 12 (2 mg/kg) (7 mg/kgtotal dose). Cisplatin was prepared daily and 2 ml of 0.9% saline wasadministered subcutaneously after each cisplatin injection. On day 14,the mechanical pain threshold was measured, and rats received vehicle(canola oil/gelatin), DCUKA (50 mg/kg), BCUKA (50 mg/kg) or gabapentin(30 mg/kg, a dose equimolar to DCUKA and BCUKA). One and two hourslater, the mechanical pain threshold was again tested. Data are reportedas the ratio of the mechanical pain threshold measured after DCUKA,BCUKA or gabapentin treatment to the baseline mechanical pain threshold(measured on the same paw). Statistical analysis was a 2-way ANOVA withrepeated measures (Proc Mixed, SAS v9.3). Treatment and time were themain fixed independent effects, and the interaction was also tested. Theanimal identification number was used as a repeated measure. Fisher'sLSD post hoc tests were used to compare significance (p<0.05) betweenthe different times within treatment groups.

Acute Effect of DCUKA on Compete Freund's Adjuvant (CFA)-InducedNeuropathic Pain. After measurement of the baseline paw withdrawalthreshold, CFA (0.1 ml) was administered subcutaneously into the plantarsurface of the left hind paw of the rat under light isofluraneanesthesia (5% for induction and 2% for maintenance). Rats were left intheir home cage for 48 hours. Paper bedding was used to avoid pressureneuropathies caused by hard bedding. At 48 or 60 hours after CFAinjection, rats were given vehicle (canola oil/gelatin) orally, orvarious doses of DCUKA orally, by intragastric gavage, and themechanical pain threshold was determined one hour later. Data arepresented as the ratio of the mechanical pain threshold measured afterDCUKA treatment to the baseline mechanical pain threshold.

Data Analysis of CFA Experiments: Acute Effect of 50 mg/kg DCUKA andMeta-Analysis of DCUKA Dose-Response. Each experiment was analyzed witha 1-way ANOVA (Proc Glm or Proc Mixed, SAS v9.3, Cary, N.C.). Treatmentgroup was the fixed independent effect tested. All models were testedfor equal variances among treatment groups (Barlett's test forhomogeneity of variances) and normality (Kolmogorov-Smirnov goodness offit test). If the data did not pass these assumptions, a mixed model wasused. All analyses used Fisher's LSD post hoc tests to comparestatistical significance (p-value<0.05) among the different treatmentgroups.

Comparison of the Effect of DCUKA and BCUKA on CFA-Induced NeuropathicPain. The baseline mechanical pain threshold was determined, and animalswere treated with CFA as described above. At 48 hours after CFAtreatment, rats were given vehicle (canola oil/gelatin) (n=17), 50 mg/kgDCUKA (n=17) or 50 mg/kg BCUKA (n=6). The mechanical pain threshold wastested one hour later, and data are reported as the ratio of the painthreshold after vehicle, DCUKA or BCUKA treatment to the baseline painthreshold, measured on the same paw. A one-way ANOVA followed byFisher's LSD post hoc test was used to determine statisticalsignificance (p<0.05).

Acute Effect of DCUKA on Streptozotocin (STZ)-Induced Neuropathic Pain(Model of Diabetic Neuropathic Pain). After measurement of the baselinepaw withdrawal threshold, baseline body weight and blood glucoseconcentrations were determined (blood glucose measured on tail blood,using the ASCENSIA CONTOUR Blood Glucose Monitoring System, Bayer,Pittsburgh, Pa.). Rats were fasted overnight and injectedintraperitoneally with vehicle (20 mM sodium citrate, pH 4.5,Sigma-Aldrich), or 50 mg/kg STZ in vehicle. The STZ solution wasprepared each day and used within 10 minutes. Food was given to the rats30 minutes after STZ treatment. Three days after STZ treatment, bloodglucose levels were again measured, and rats with a blood glucose levelabove 350 mg/dl were considered “diabetic”. If the blood glucose levelwas below 350 mg/dl, the rat was given a second dose of STZ (45 mg/kg),using the same procedure. Fourteen days after the first STZ treatment,rats were given vehicle (canola oil/gelatin) or various doses of DCUKAorally, and the mechanical pain threshold was tested at various timesafter these treatments. Data are presented as the ratio of themechanical pain threshold following DCUKA treatment to the baselinemechanical pain threshold (measured on the same paw).

Individual Experimental Analysis. Data points were considered asoutliers and removed from the dataset if the ratio of the mechanicalpain threshold to baseline was outside the corresponding treatment groupmean±2 standard deviations. Each experiment was analyzed with a 1-wayANOVA (Proc Glm or Proc Mixed, SAS v9.3, Cary, N.C.). Treatment groupwas the fixed independent effect tested. All models were tested forequal variances among treatment groups (Barlett's test for homogeneityof variances) and normality (Kolmogorov-Smirnov goodness of fit test).If the data did not pass these assumptions, a mixed model was used. Allanalyses used Fisher's LSD post hoc tests to compare statisticalsignificance (p-value <0.05) between the different treatments.

FIG. 5 illustrates that DCUKA (50 mg/kg) treatment reverses neuropathicpain caused by treatment of rats with the cancer chemotherapeutic agent,cisplatin. Combined results from six experiments are shown. In allexperiments, rats were tested for baseline mechanical pain thresholdprior to cisplatin treatment. Following cisplatin treatments describedearlier, rats were given DCUKA, and one hour later, the mechanical painthreshold was again determined. The results show the ratio of themechanical pain threshold measured at one hour after vehicle or DCUKAadministration compared to the baseline (pre-cisplatin treatment)mechanical pain threshold.

FIG. 6 graphically compares the effect of DCUKA (50 mg/kg), BCUKA (50mg/kg) and gabapentin (Neurontin, 30 mg/kg) on neuropathic pain inducedby the chemotherapeutic agent, cisplatin. Rats were tested for baselinemechanical pain threshold prior to cisplatin treatment, and were treatedwith cisplatin as described earlier. Following cisplatin treatment themechanical pain threshold was measured, and rats were given oral dosesof vehicle, DCUKA, BCUKA, or gabapentin. The mechanical pain thresholdwas again measured at 1 and 2 hours after these treatments. Data are theratio of the mechanical pain threshold measured prior to DCUKA, BCUKA orgabapentin administration, and 1 and 2 hours later. Cisplatin treatmentalone (“pretreatment”) significantly reduced the mechanical painthreshold, compared to baseline, and DCUKA and BCUKA significantlyreversed the drop in mechanical pain threshold. Gabapentin, at a doseequimolar to DCUKA and BCUKA, did not significantly reverse thecisplatin-induced decrease in the mechanical pain threshold.

FIG. 7 graphically illustrates that DCUKA reverses the neuropathic paininduced by treatment of rats with Complete Freund's Adjuvant (CFA) toproduce an inflammatory response. Data are combined from threeexperiments. In each experiment, the baseline mechanical pain threshold(force, in g, causing paw withdrawal) was first measured using anelectronic von Frey anesthesiometer. Rats were then injected with 0.1 mlcomplete Freund's Adjuvant (CFA) into the plantar surface of the lefthind paw. Forty-eight to 60 hours later, when CFA-induced pain haddeveloped, rats received oral administration of vehicle (gelatin/canolaoil emulsion) or 50 mg/kg DCUKA. One hour later, mechanical painthreshold was again measured. The results show the ratio of mechanicalpain threshold measured one hour after vehicle or DCUKA treatment to thebaseline mechanical pain threshold. CFA treatment reduced the mechanicalpain threshold by approximately 60%, and DCUKA treatment reversed thiseffect and increased the mechanical pain threshold in the CFA-treatedpaw to a level not significantly different from the baseline level.

FIG. 8 graphically illustrates a comparison of the effect of DCUKA (50mg/kg) and BCUKA (50 mg/kg) to reverse neuropathic pain produced by CFAtreatment. Baseline mechanical pain threshold was measured, and ratswere treated with CFA as described earlier. Forty-eight hours later,rats were given vehicle (canola oil/gelatin), DCUKA or BCUKA, and onehour later the mechanical pain threshold was measured. CFA treatmentreduced the mechanical pain threshold to about 40% of baseline, andtreatment with DCUKA (n=17) or BCUKA (n=6) reversed the mechanical painthreshold to a level not significantly different from baseline.

The dose dependence of the effects of DCUKA on neuropathic pain producedby CFA was determined using a meta-analysis approach. The results areshown in FIG. 9. In all experiments with CFA, the baseline mechanicalpain threshold was measured with an electronic von Frey anesthesiometer.CFA was injected into the plantar surface of the left hind paw and, at48 hours after injection, rats were given oral vehicle (gelatin/canolaoil) or DCUKA. The mechanical pain threshold was again measured at 60min after vehicle or DCUKA administration. For an experiment to beincluded in the meta-analysis, the requirements were: 1) CFA treatmentproduced at least a 25% decrease in the mechanical pain threshold; 2)the pain threshold was measured at 60 min after DCUKA or vehicleadministration. Five experiments, in which different doses of DCUKA weretested, met these requirements. The mechanical pain threshold, as aratio to the baseline mechanical pain threshold, is shown as mean±SEM.There was a significant overall effect of DCUKA (F(5,125)=7.71,P<0.0001). CFA treatment reduced the mechanical pain threshold byapproximately 60%, and this effect was significantly reversed by DCUKAdoses of 30 mg/kg and higher, i.e., the pain threshold returned to thebaseline level.

FIG. 10 illustrates that DCUKA reverses the neuropathic pain thataccompanies diabetes. Diabetes is induced in rats by injection ofstreptozotocin (STZ) as described earlier. Combined data from threeexperiments are shown. In each experiment, baseline mechanical painthreshold was tested using an electronic von Frey anesthesiometer.Fourteen days after STZ treatment, vehicle (gelatin/canola oil) or 40 or50 mg/kg DCUKA (these doses did not have significantly differenteffects) was administered orally and mechanical pain was assessed 90minutes later. The results show the ratio of the mechanical painthreshold following vehicle/DCUKA treatment to the baseline mechanicalpain threshold. STZ reduced the mechanical pain threshold byapproximately 40%, and this effect was reversed to the baseline level byDCUKA treatment.

The dose-dependence of the effects of DCUKA on STZ-induced neuropathicpain was determined by a meta-analysis approach. Requirements for anexperiment to be included in the meta-analysis were: 1) STZ treatmentinduced neuropathic pain, as measured by a decrease in mechanical painthreshold of at least 25%; 2) pain was measured 90 minutes after vehicleor DCUKA treatment. Four experiments that met these criteria wereincluded in the meta-analysis.

FIG. 11 shows the dose-dependence of the effect of DCUKA to reduceSTZ-induced neuropathic pain. The data are reported as the ratio of themechanical pain threshold after vehicle or DCUKA treatment to thebaseline pain threshold. There was an overall significant effect ofDCUKA on mechanical pain threshold (F(7, 115=8.48, p<0.0001). STZtreatment induced approximately a 40% reduction in the pain threshold,and doses of DCUKA of 30 mg/kg and higher reversed the effect of STZ andincreased the pain threshold back to the baseline level.

Example 6 Naltrindole Reversal of the Effect of DCUKA on CFA-InducedNeuropathic Pain

This example demonstrates that the anti-allodynic response toadministration of DCUKA can be reversed by introducing naltrindole (adelta opiate receptor antagonist) to the site of inflammation.

Rats were tested for baseline mechanical pain threshold (von Frey test)and given an intraplantar injection of Complete Freunds Adjuvant (CFA)in the left hind paw. After two days, rats were treated with eithervehicle or 50 mg/kg DCUKA, \orally, and 45 min after DCUKA/vehicletreatment, rats received either Naltrindole (10 μg per 50 μl per paw) orsaline (50 μl per paw) s.c., in the plantar surface of left paw. At 15minutes after Naltrindole or saline injection, the pain threshold wasassessed.

FIG. 12 illustrates that the presence of naltrindole at the site ofinflammation reversed the anti-allodynic effect of DCUKA. CFA treatmentreduced the mechanical pain threshold by about 50%, and DCUKA/salineincreased the pain threshold back to baseline level. In the presence ofnaltrindole and DCUKA, the allodynic response was no different from thatin animals injected with just the vehicle, and naltrindole had no effecton its own. These results underscore the importance of delta opiatereceptors in the actions of DCUKA, in vivo, as was indicated by the invitro binding and functional assays.

Example 7 Prevention of Cisplatin- and CFA-Induced Neuropathic Pain byDCUKA

This example illustrates that treatment of animals with DCUKA followinginsult, but prior to development of neuropathic pain, can prevent thedevelopment of the pain. Rats were treated with cisplatin or CFA, andmechanical pain threshold was measured, as described under Example 5.

Prevention of CFA-Induced Neuropathic Pain by DCUKA.

After baseline measurement of the mechanical pain threshold, rats weretreated with CFA as described earlier. Following the CFA injection, ratswere given vehicle (canola oil/gelatin) (n=7) or 50 mg/kg DCUKA (n=7)orally by intragastric gavage. Rats then received three more treatmentswith vehicle or DCUKA at 12-hour intervals. At 60 hours after CFAtreatment, the mechanical pain threshold was again tested.

Prevention of Cisplatin-Induced Neuropathic Pain by DCUKA. One day afterbaseline measurement of the mechanical pain threshold, rats wereinjected i.p. with cisplatin. Cisplatin injections were again given ondays 4, 8, and 12. Starting one hour after the first cisplatininjection, rats were given 50 mg/kg DCUKA or vehicle via intragastricgavage twice daily for 14 days. After the last treatment (day 15) themechanical pain threshold was again tested. The pain threshold was thentested once weekly for 4 weeks, with no further treatments.

FIG. 13 shows that DCUKA can prevent the development of neuropathic paincaused by CFA. Data show the ratio of the mechanical pain threshold at60 hours after CFA treatment to the pre-CFA baseline mechanical painthreshold. CFA treatment significantly reduced the pain threshold byapproximately 70%. However, when animals were given DCUKA daily prior totesting, the threshold was not reduced (P=0.16), and remained close tothe baseline pain threshold, i.e., DCUKA has the ability to prevent thedevelopment of inflammatory pain, when given after administration of aninflammatory agent.

FIG. 14 illustrates that repeated DCUKA treatments during the periodbetween the administration of cisplatin and pain testing, prevented thedevelopment of pain in the rat cisplatin-induced neuropathic pain model.Data are presented as mean±SEM of the mechanical pain threshold (pawwithdrawal threshold). Injection of Cisplatin resulted in a significantdecrease in mechanical pain threshold over days in the rats treatedchronically with vehicle. With repeated DCUKA treatments, there was nostatistically significant decrease of pain threshold compared to thepre-cisplatin baseline. The results show that DCUKA has the ability toprevent the development of chemotherapy-induced neuropathic pain, whengiven after the administration of the chemotherapeutic agent.

Example 8 DCUKA and DCUK-OEt Enhance the Effect of Morphine onCFA-Induced Neuropathic Pain

Example 8 demonstrates that combining low doses of DCUKA and morphineresults in a synergistic effect on reduction of mechanical allodynic andthermal hyperalgesic pain produced by an inflammatory agent.

CFA treatment and measurement of mechanical pain threshold are describedunder Example 5. In this experiment, mechanical pain threshold wastested at baseline. At 48 hours after CFA treatment, vehicle, DCUKA ormorphine, or the combination of DCUKA and morphine, were injected 30minutes prior to measurement of the mechanical pain threshold.

Thermal Hypersensitivity Test (Radiant Heat Paw Withdrawal Test). Ratswere placed in clear plastic chambers on a glass surface and werehabituated for 15 minutes before testing. Thermal sensitivity wasmeasured by using paw withdrawal latency to a radiant heat stimulus. Aradiant heat source (i.e., infrared) was activated with a timer andfocused onto the plantar surface of the left hind paw. A motion detectorthat halted both lamp and timer when the paw was withdrawn determinedpaw withdrawal latency. The latencies were measured before and afterdrug or vehicle administration. A maximum cutoff of 33 seconds was usedto prevent tissue damage. In this experiment, rats were injected withDCUK-OEt, immediately followed by morphine at increasing dose ratios,and tested 30 minutes later.

FIG. 15, Panel A, demonstrates that combining doses of DCUKA andmorphine, that in themselves, are ineffective in producing analgesia,results in a complete reversal of inflammation-induced chronic pain.FIG. 15, Panels B and C, provides evidence that the combination ofDCUK-OEt and morphine provides a supra-additive effect, as compared toeither agent given alone. In this case, thermal hyperalgesia produced byCFA treatment was tested. The ED₅₀ for the anti-hyperalgesic response tomorphine was significantly reduced by administering DCUK-OEt in a doseratio to morphine of >30:1. For instance, a dose of 0.2 mg/kg morphineproduces approximately a 20% antihyperalgesic response. Combined with6.4 mg of DCUK-OEt, the response is increased to 40%.

Example 9 DCUKA Enhances the Effect of Aspirin on STZ-InducedNeuropathic Pain

Example 9 demonstrates that in another model of chronic pain, the STZdiabetic neuropathy model, DCUKA can synergize with another analgesic(aspirin) to reduce allodynia.

After administration of streptozotocin (STZ) as described in Example 5,rats were tested for allodynia using a von Frey apparatus. Sixty minutesprior to testing, rats were divided into four groups. Group 1 receivedvehicle; group 2 received DCUKA; group 3 received aspirin; and group 4received a combination of DCUKA and aspirin.

FIG. 16 demonstrates that doses of DCUKA (12.5 mg/kg) or aspirin (25mg/kg) which by themselves do not have any significant effect onallodynia, when combined, completely reverse the hyper-responsiveness,i.e., return the pain threshold to the baseline level.

Example 10 Effect of DCUK-OEt on “Relapse” Alcohol Consumption byAlcohol-Dependent Rats

This example illustrates the use of an animal model of alcoholdeprivation in alcohol dependent animals to produce relapse drinking,and shows that DCUK-OEt can prevent relapse in those animals.

This model is thoroughly described in a paper by (Spanagel and Holter,1999). The model consists of a conditioning phase where individuallyhoused male Wistar rats are allowed ad libitum access to food and 0, 5,10 and 20% alcohol solutions in a four-bottle choice paradigm. The ratsare weighed every 3^(rd) and 4^(th) day and food and fluid consumptionis measured. The positions of bottles are changed once a week and aftertwo months of continuous alcohol access, rats are deprived of alcoholfor two weeks. After the deprivation phase, all alcohol solutions arepresented again for 5 weeks and the procedure is repeated for ˜1 year.At this point an alcohol deprivation phase is instituted.Pre-deprivation (baseline) drinking is recorded for 3-4 days and thenall alcohol is taken from the animals and they are only given water todrink for two weeks. Following alcohol deprivation, alcohol intake(total grams of ethanol/kg body weight) and water intake (ml/kg bodyweight) are measured daily. The alcohol preference ratio is calculatedas (ethanol consumption [g/kg])/(water consumption [ml/kg]).

For this experiment, animals were divided into groups of 6-8 rats suchthat the mean baseline total alcohol intake was approximately the same(˜2.8 g/kg/day) across all groups. Baseline drinking was monitoredfollowed by 14 days of alcohol deprivation. Each animal then receivedi.p. injections of DCUK-OEt twice daily at 12 hour intervals starting at7 PM prior to the reintroduction of ethanol for a total of 3 (20 mg/kg)or 5 injections (75 mg/kg). Acamprosate, an already marketed agent forreducing relapse, was used as a positive control and four doses of 50mg/kg or 200 mg/kg were injected over a two-day period. Alcohol bottleswere re-introduced after the first (acamprosate) or second (DCUK-OEt)injection.

The results in FIG. 17 demonstrate that a 20 mg/kg dose of DCUK-OEtproduced a transient, but significant, reduction in relapse drinking,similar to a larger dose of acamprosate (50 mg/kg). A 75 mg/kg dose ofDCUK-OEt produced an almost complete abolition of alcohol intake eventhough the animals had alcohol available in a free-choice situation.This reduction in drinking was evident even several days after theadministration of DCUK-OEt was stopped. The reduction of ethanolconsumption was compensated by an increased consumption of water bythese animals, greatly reducing the preference ratio (data not shown).By comparison to acamprosate, the effects of DCUK-OEt were greater inmagnitude and were longer lasting even though the dose of DCUK-OEt wasmore than two fold lower overall. This indicates that DCUK-OEt would bean effective medication in preventing/reducing relapse drinking inaddicted individuals.

Example 11. Effect of DCUK-OEt on Operant Responding for Alcohol byAlcohol Dependent Rats

This example illustrates that ethanol dependent rats are motivated towork for ethanol reward during a period of forced abstinence and thatDCUK-OEt reduces responding for the alcohol reward.

The methods used in this study are extensively described in (Vendruscoloet al., 2012). The rats were first trained to self-administer ethanol inan operant chamber. The rats were given free-choice access to alcohol(10% w/v) and water for 1 day in their home cages to habituate them tothe taste of alcohol. The rats were then subjected to an overnightsession in the operant chambers with access to one lever that deliveredwater (FR1). Food was available ad libitum during this training. After 1day off, the rats were subjected to a 2 hour session (FR1) for 1 day anda 1 hour session (FR1) the next day, with one lever delivering alcohol.All of the subsequent sessions lasted 30 minutes, and two levers wereavailable (left lever: water; right lever: alcohol). Once trained, therats were made dependent by chronic, intermittent exposure to alcoholvapors (Gilpin et al., 2008). They underwent cycles of 14 hours ofethanol exposure (blood alcohol levels during vapor exposure rangedbetween 150 and 250 mg %) and 10 hours in the absence of ethanol vapor,during which behavioral testing for acute withdrawal occurred (i.e., 6-8hours after vapor was turned off, when brain and blood alcohol levelswere negligible). Nondependent rats were those not exposed to alcoholvapor. Pharmacological testing occurred after at least 2 months of vaporexposure, when full dependence was observed. DCUKA-OEt was administeredi.p. at the doses specified in FIG. 18, sixty minutes prior to placingthe animal in the operant chamber. The vehicle for DCUK-OEt was injectedin certain animals as a control.

FIG. 18 illustrates the effects of DCUK-OEt and DCUKA on alcoholself-administration in ethanol dependent rats exposed to either ethanol(alcohol) vapor or water vapor. The data in FIG. 18 demonstrate thatDCUKA has no significant effect on repressing alcoholself-administration compared to baseline (BSL) in ethanol vapor-exposedrats, while DCUK-OEt at 20 mg/kg brought the ethanol vapor exposed ratsdown to the same level of self-administration as non-ethanol pretreatedcontrol rats. Higher doses of DCUK-OEt (35 and 50 mg/kg) reduced leverpressing for ethanol to even lower levels in the alcohol-dependent rats.None of the doses of DCUK-OEt tested had any effect on water intake.

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Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method of treating or preventing alcoholaddiction relapse in a subject in need thereof, the method comprisingadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of a compound of Formula (A) or apharmacologically acceptable salt thereof, and a pharmaceuticallyacceptable carrier; wherein the compound of Formula (A) is:


2. The method of claim 1, wherein the therapeutically effective amountof the compound of Formula (A) is from about 1 mg/kg of body mass toabout 200 mg/kg of body mass.
 3. The method of claim 1, wherein thesubject is abstinent from alcohol.
 4. The method of claim 1, wherein thepharmaceutical composition is a tablet, a capsule, a solution, asuspension, or an aerosol.
 5. A method of treating pain in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of a pharmacologically acceptabletoluene sulfonic acid salt of a compound of Formula (IV):

wherein: R⁷ is alkyl, cycloalkyl, aminoalkyl, or phenyl; wherein each isoptionally substituted one or more times with an alkyl group or analkyloxy group; R⁸ is H, alkyl, cycloalkyl, aminoalkyl, or phenyl;wherein each is optionally substituted one or more times with an alkylgroup or an alkyloxy group; E¹ is —C(═O)OR⁹, —C(═O)R⁹, —C(═O)N(R⁹)₂, or(C(R⁹)₂)_(n)—OR⁹; n is 1, 2, 3, or 4; R⁹ is H, C₁-C₄ alkyl, or C₁-C₄alkyl substituted with one or more moiety selected from the groupconsisting of C₁-C₄ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, halo,heteroaryl, Z⁵R²⁴, and N(R²⁵)(R²⁶); Z⁵ is O, S, C(═O)O or O—C(═O); R²⁴,R²⁵ and R²⁶ are each independently C₁-C₄ alkyl; and X² and X³ are eachindependently halogen or nitro.
 6. The method of claim 5, wherein thepain is chronic pain.
 7. The method of claim 5, wherein the pain isneuropathic pain.
 8. The method of claim 5, wherein the pain isperipheral neuropathic pain.
 9. The method of claim 5, wherein thetherapeutically effective amount is from about 1 mg/kg of body mass toabout 200 mg/kg of body mass.
 10. The method of claim 5, wherein: R⁷ isalkyl, cycloalkyl, aminoalkyl, or phenyl; R⁸ is H, alkyl, cycloalkyl,aminoalkyl, or phenyl; E¹ is —C(═O)OR⁹ or —C(═O)R⁹; n is 1, 2, 3, or 4;R⁹ is H, C₁-C₄ alkyl; and X² and X³ are each independently halogen ornitro.
 11. The method of claim 5, wherein the compound of Formula (IV)is a compound of Formula (B):


12. The method of claim 11, wherein the pain is chronic pain.
 13. Themethod of claim 5, further comprising administering to the subject anopioid agonist, a non-steroidal anti-inflammatory drug, acorticosteroid, a serotonin reuptake inhibitor, a calcium channelblocker, an anticonvulsant, or a combination of two or more thereof. 14.The method of claim 5, further comprising administering to the subjectmorphine, oxycodone, aspirin, ibuprofen, naproxen, duloxetine,fluoxetine, gabapentin, clonidine, or pregabalin.
 15. A method oftreating pain in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising a pharmacologically acceptabletoluene sulfonic acid salt of a compound of Formula (B) and apharmaceutically acceptable carrier; wherein the compound of Formula (B)is:


16. The method of claim 15, further comprising administering to thesubject an opioid agonist, a non-steroidal anti-inflammatory drug, acorticosteroid, a serotonin reuptake inhibitor, a calcium channelblocker, an anticonvulsant, or a combination of two or more thereof. 17.The method of claim 15, further comprising administering to the subjectmorphine, oxycodone, aspirin, ibuprofen, naproxen, duloxetine,fluoxetine, gabapentin, clonidine, or pregabalin.
 18. The method ofclaim 15, wherein the pain is chronic pain.
 19. The method of claim 15,wherein the pain is neuropathic pain.
 20. The method of claim 15,wherein the pain is peripheral neuropathic pain.